i

ABSTRACT

DEVELOPMENT OF GRAFTING PROTOCOLS FOR SALT-TOLERANT CACTUS PEAR (OPUNTIA FICUS-INDICA)

Protocols for the production of grafted salt-tolerant cactus pear (Opuntia

ficus-indica) were developed as follows:

Four different surface sterilization protocols and four different shoot

induction media were tested for the establishment of sterile cultures of three

patented salt-tolerant cactus pear (Opuntia ficus-indica) cultivars grown in central

California. 6-Benzylaminopurine (BA) levels were varied to assess the effect of

BA concentration on shoot induction in one of the media. A harsh protocol and a

gentle protocol both provided adequate surface sterilization. Media with 5mg/l BA

(El Finti, El Boullani, El Ayadi, Ait Aabd, & El Mousadik, 2012) and 2.5 mg/l BA

(Estrada-Luna, 1988) were the most effective at shoot induction.

Shoot proliferation, root induction and in vitro grafting were examined in

three cultivars of salt-tolerant cactus pear for the development of an in vitro

production system. Lower concentrations of BA in shoot proliferation media were

shown to eliminate the production of abnormal phenotype explants (p = 0.028). In

vitro grafting results were inconclusive because all grafts showed death of

rootstock, death of scion, or contamination.

Parallel Wedge Grafts, Horizontal Grafts, and Perpendicular Wedge Grafts

were tested in two different scion/rootstock combinations of salt-tolerant cactus

pear. Perpendicular Wedge Grafting was the most successful of the three

techniques with a success rate of 66.67%. Overall, a successful in situ grafting

protocol was developed and progress was made in the development of an in vitro

production system.

Jacob Hurst December 2016

ii

i

DEVELOPMENT OF GRAFTING PROTOCOLS FOR SALT-

TOLERANT CACTUS PEAR (OPUNTIA FICUS-INDICA)

by

Jacob Hurst

A thesis

submitted in partial

fulfillment of the requirements for the degree of

Master of Science in Plant Science

in the Jordan College of Agricultural Sciences and Technology

California State University, Fresno

December 2016

ii

© 2016 Jacob Hurst

iii

APPROVED

For the Department of Plant Science:

We, the undersigned, certify that the thesis of the following student meets the required standards of scholarship, format, and style of the university and the student's graduate degree program for the awarding of the master's degree. Jacob Hurst

Thesis Author

John Bushoven (Chair) Plant Science

Dave Goorahoo Plant Science

Gary Bañuelos USDA-ARS Parlier

For the University Graduate Committee:

Dean, Division of Graduate Studies

iv

AUTHORIZATION FOR REPRODUCTION

OF MASTER’S THESIS

X I grant permission for the reproduction of this thesis in part or in

its entirety without further authorization from me, on the

condition that the person or agency requesting reproduction

absorbs the cost and provides proper acknowledgment of

authorship.

Permission to reproduce this thesis in part or in its entirety must

be obtained from me.

Signature of thesis author:

v

ACKNOWLEDGMENTS

I thank Dr. John Bushoven for serving as my academic advisor and the

chair of my thesis committee. I thank Dr. Gary Bañuelos for being my research

mentor and a member of my thesis committee, and for allowing me unhindered

access to his patented Opuntia ficus-indica material. I also thank Dr. Dave

Goorahoo for serving on my thesis committee and for providing support and

advice during the planning and execution of my research projects.

I thank Irvin Arroyo for showing me the experimental field site, for

assisting with cladode harvest, and for providing Opuntia cultivation advice. I

thank Jerry Serimian for his advice on in situ grafting and for sharing his

knowledge about the Horizontal grafting technique.

I thank Calliope Correia for providing me with greenhouse space, pots, and

potting soil. I thank Eshan Bhardwaj for providing invaluable help in the lab with

media preparation and establishment of sterile cultures, and for providing sound

advice on sterilizing agents. I thank Mauro Trujillo and Brian Coelho for help with

initial in vitro work.

I also thank my parents, Mark and Linda Hurst, for their love and support

(both emotional and financial) throughout this endeavor.

vi

TABLE OF CONTENTS

Page

LIST OF TABLES ................................................................................................ viii

JUSTIFICATION OF STUDY AND LITERATURE REVIEW ............................ 1

Statement of Problem ........................................................................................ 1

Justification and Significance of Study ............................................................. 1

Literature Review .............................................................................................. 4

CHAPTER 1: ESTABLISHMENT OF ASEPTIC CULTURES AND SHOOT INDUCTION IN THREE CULTIVARS OF SALT-TOLERANT CACTUS PEAR (OPUNTIA FICUS-INDICA) .......................................... 15

Introduction ..................................................................................................... 15

Materials and Methods .................................................................................... 15

Results ............................................................................................................. 22

Discussion ....................................................................................................... 25

CHAPTER 2: IN VITRO SHOOT PROLIFERATION, ROOTING AND MICROGRAFTING OF THREE CULTIVARS OF SALT-TOLERANT CACTUS PEAR (OPUNTI FICUS-INDICA) ............................................. 28

Introduction ..................................................................................................... 28

Materials and Methods .................................................................................... 29

Results ............................................................................................................. 30

Discussion ....................................................................................................... 31

CHAPTER 3: IN SITU GRAFTING OF SALT-TOLERANT CACTUS PEAR (OPUNTIA FICUS-INDICA) ...................................................................... 33

Introduction ..................................................................................................... 33

Materials and Methods .................................................................................... 33

Results ............................................................................................................. 37

Discussion ....................................................................................................... 40

CHAPTER 4: CONCLUSIONS ............................................................................. 41

Page

vii vii

In Situ Grafting ................................................................................................ 41

In Vitro Production and Grafting .................................................................... 42

REFERENCES ....................................................................................................... 44

viii

LIST OF TABLES

Page

Table 1 Summary of Experimental Conditions..................................................... 21

Table 2 Contamination and Shoot Induction Rates by Sterilization Method, Media, Origin and Age of Cladodes Used for Initiation of Sterile Cultures. Shoot Initiation Rates Calculated for Clean Explants of Patented Cultivars Only .......................................................................... 23

Table 3 Shoot Proliferation and Incidence of Normal and Abnormal Phenotypes .............................................................................................. 31

Table 4 Root Production by Phenotype ................................................................ 31

Table 5 Graft Failure by Type .............................................................................. 31

Table 6 Success Rates for Three Grafting Techniques ........................................ 40

ix

LIST OF FIGURES

Page

Figure 1. Ideal in vitro production system ............................................................... 3

Figure 2. Ideal in situ production system ................................................................. 3

Figure 3. Explants, shoot initiation, growth, and proliferation .............................. 18

Figure 4. Examples of fungal contamination arising on and around the areole .... 23

Figure 5. Parallel wedge graft progression from graft initiation to graft failure ... 35

Figure 6. Completed horizontal grafts ................................................................... 38

Figure 7. Initial stage of perpendicular wedge graft and successful

perpendicular wedge graft ...................................................................... 39

Figure 8. A production system for field-grafted plants ......................................... 41

1

JUSTIFICATION OF STUDY AND LITERATURE REVIEW

Statement of Problem

The drought in California from 2012-2014 was the worst in recorded

history and, based on tree-ring data, was the most severe period of drought in the

past 1200 years (Griffin & Anchukaitis, 2014). Unfortunately, drought is predicted

to become more frequent and more severe in the coming decades (Diffenbaugh,

Swain, & Touma, 2015). Drought in California has a pronounced negative effect

on irrigated agriculture in the western San Joaquin Valley. Farmers there rely on

surface water to grow crops and importantly, to leach naturally-occurring salts

from their soil. With a shortage of water, the range of crops that can be grown is

severely limited and farmers are forced to fallow large tracts of land because most

conventional crops are relatively salt-sensitive and few salt-tolerant alternatives

are available (Glenn, Brown, & Blumwald, 1999).

Justification and Significance of Study

In response to these conditions, researchers with the USDA-ARS Parlier

and California State University, Fresno have developed the “Seleno” series of salt-

tolerant prickly pear cactus (Opuntia ficus-indica) (Banuelos, Freeman, & Diener,

2013a-d). This alternative crop can be grown on land that would be unsuitable for

other crops and produces unique antioxidant-rich fruit with potential anti-

carcinogenic properties (Banuelos et al., 2012) when grown on high-selenium soils

such as those in the western San Joaquin Valley. Growing this crop could allow

farmers to produce a value-added crop on land that might otherwise be left out of

production. There are four varieties in the series, named for the color of their fruit

(Seleno-Red, -Green, -Orange, and -Purple). Seleno-Orange and -Green are more

tolerant of saline conditions than the other two varieties, but their fruit are less

desirable than the larger, more globular red or purple fruit from Seleno-Red and

2 2

-Purple. Can Seleno-Red or -Purple be grafted onto Seleno-Green or -Orange to

allow for the production of desirable fruits under poor growing conditions?

Cacti have been grafted for many years, but mainly in ornamental varieties

that display a columnar growth habit. Opuntia species have not been widely

grafted for two reasons: desiccation and pathogenic fungal growth (Maldonado &

Zapien-Barragan, 1977). Opuntia spp. cladodes are flat and wide, which increases

the surface area exposed to the air relative to a cylindrical stem. This physical

characteristic leads to greater water loss and increased risk of exposure to

pathogens when the cladodes are grafted. An optimized grafting process should

reduce the negative impacts of desiccation and fungal pathogens and subsequently

allow for the production of grafted salt-tolerant O. ficus-indica plants. These

grafted plants could be a new crop for use in poor quality soils on the West Side of

the San Joaquin Valley. The purpose of this study is to develop systems for the

production of grafted cactus plants in vitro and in situ.

In an ideal in vitro production system (Figure 1), plant material would be

sterilized and then go through initial shoot induction. Thereafter, in vitro shoot

proliferation would provide a continuous supply of sterile rootstock and scion

material for in vitro grafting and allow for rapid production of grafted plants that

would be acclimated to ex vitro conditions and eventually moved to the field.

An ideal in situ production system schematically looks very similar to the

in vitro system (Figure 2), but skips sterilization and acclimation stages because it

all occurs ex vitro. Perhaps the most important difference is that the in situ system

can only be utilized during periods of active growth and stops during the winter,

while the in vitro system can be implemented year-round. However, larger plants

can be grafted in situ, which could have implications for survival in the field

(Turner, Alcorn, & Olin, 1969).

3 3

Figure 1. Ideal in vitro production system

Figure 2. Ideal in situ production system

4 4

Literature Review

The Physiology of Grafting: Graft Union Formation and Rootstock-Scion Interactions

Grafting is a well-established technique of plant propagation that has been

practiced at least as far back as 1560 BC (Mudge et al., 2009). The reasons that

justify grafting are many, but the central appeal of grafting is that it allows for the

combination of traits from two or more types of plants in a relatively short time

without the need to breed all the traits into a single plant. This technique allows for

the rapid production of specimens that are difficult to replicate through traditional

breeding and it makes it possible for certain grafted crops to thrive under

conditions that would normally have a negative impact on yield (Hartmann,

Kester, Davies, & Geneve, 2010). Grafting is conceptually straightforward, but

physiologically is a complicated process that merely begins with the physical act

of placing a scion on a rootstock. This paper will review literature relevant to the

physiology of graft union formation and the subsequent interactions between

rootstock and scion.

Graft union formation. Graft union formation is generally divided into five

stages: 1) lining up vascular cambiums of the rootstock and scion; 2) wounding

response; 3) callus bridge formation; 4) differentiation of vascular cambium across

the callus bridge, and 5) production of secondary xylem and phloem (Hartmann et

al., 2010). First, the vascular cambiums need to be lined up between the rootstock

and scion to maximize contact. Next, the damaged cells on the cut ends of the

rootstock and scion die and form a necrotic layer. This layer of dead cells contains

pectin and other sticky compounds from the cytoplasm, which act like glue and

may help keep the graft union moist (Moore & Walker, 1981a,b). Eventually, the

5 5

material from the necrotic layer is absorbed back into the living cells and the

proliferation of undifferentiated callus begins. Much like the construction of the

Transcontinental Railroad, the rootstock and scion begin forming callus at their

respective ends of the graft junction and then meet in the middle to complete the

callus bridge (Williams, 1996). After the callus forms a continuous line, a similar

ends-to-middle differentiation of xylem and phloem initials begins in the callus

cells. Once the xylem and phloem initials from the rootstock and scion come into

contact, the vascular cambiums are connected and secondary xylem and phloem

begin to form. Estrada-Luna, Lopez-Peralst, and Cardenas-Soriano (2002) include

healing of the epidermis as an additional final stage in graft union formation. It is

important to consider this step for practical settings because the epidermis serves

as the main barrier to pathogens, and therefore the vascular system is vulnerable to

infection until the exterior of the wound is sealed. Their study of Opuntia spp.

shows that under ideal conditions, the process of graft union formation takes

around 30 days from grafting to a successfully healed graft union (Estrada-Luna et

al., 2002).

Factors Affecting Rate of Successful Graft Union Formation

Provided that grafting is conducted with disease-free materials, there are

four main factors that can have an impact on successful formation of the graft

union: cambial layer contact, temperature, plant hormones, and incompatibility of

the two species being grafted. Each of these reasons will be described below.

Cambial layer contact. As previously mentioned, the vascular cambiums of

the rootstock and scion need to be appropriately aligned for graft union formation

to proceed. Appropriate alignment can mean different things in different cases. If

6 6

the rootstock and scion are of different diameters, proper alignment may involve

only one side of the vascular cambiums (Hartmann et al., 2010). To maximize the

likelihood of successful graft union formation, the cambial layers of the rootstock

and scion should have the highest degree of contact possible. Shimomura and

Fujihara (1978) showed that while graft unions successfully formed even when the

rootstock and scion vasculatures were misaligned by 1 mm, only 13% of plants

grafted in this manner formed successful graft unions. Plants that were grafted

with direct contact between the rootstock and scion vascular bundles had virtually

100% successful graft union formation (Shimomura & Fujihara, 1978).

Temperature. Temperature affects the success of graft union formation by

regulating the speed of metabolic processes within the rootstock and scion.

Extreme temperatures can also damage cells or proteins. Generally, warmer

temperatures increase the speed of graft union formation, while cooler

temperatures can delay the process (Hartmann et al., 2010). If conditions get too

hot or too cold, graft union formation will not be successful. In black walnut

(Juglans nigra), Sitton (1931) demonstrated that the optimal temperature for graft

union formation is around 75 to 80º F.

Hormones. Plant hormones play an important role in almost every aspect of

plant growth and development. The two hormones that play an important role in

the formation of graft unions are auxins and cytokinins, with the majority of

research focusing on auxins.

Auxin (IAA, or indole acetic acid) that is produced in shoot apices exhibits

a one-way basipetal flow down to the roots (Aloni, Cohen, Karni, Aktas, &

Edelstein, 2010). This flow is the product of mixed passive and active transport of

auxin molecules within plant cells. Auxin enters a cell through diffusion or active

7 7

transport and is held inside the cell by acid trapping, in which the pH of the

cytoplasm causes auxin molecules to deprotonate and lose their ability to pass

through cell membranes. Auxin then travels via basipetal transport. Once the

auxin reaches the basipetal region of the cell, active transport proteins send the

hormone out into the top of the cell below, where the process begins anew until

auxin reaches the roots. Auxin is typically associated with root formation and

differentiation of vascular tissues.

Cytokinins are generally produced in the roots and are translocated up to

shoots (Aloni et al., 2010). They are typically associated with shoot initiation and

cell division. Cytokinins are involved in budbreak after dormancy and encourage

branching by activating lateral buds.

Hormone applications for grafting. Applying hormones to graft junctions or

(in the case of auxins) to the tip of the scion material has been shown to influence

graft union formation success rates. In the Shimomura and Fujihara cactus study

(1978), the successful graft union formation rate was improved by applications of

auxin even in treatments where there was less-than-optimal rootstock-scion

cambial contact. Cummins (1997) demonstrated that Malus trees treated with

auxin at the time of grafting were more likely to successfully form a graft union

and reach a saleable size during the next season than their untreated counterparts.

Similar results have been obtained in cactus. Moghadam, Ardebili, and Rezaie

(2014) showed that applications of indole-3-butyric acid (IBA) could improve the

number of successful grafts in an intergeneric rootstock/scion combination. The

hormone applications also improved the health and vigor of the grafted plants.

Measures of vigor, such as number of buds, diameter of scion, thickness of

cambial layer, and number of activated buds, were all improved when grafted

8 8

plants received three applications of 100 ppm IBA over the course of their study.

Lower measures of vigor were exhibited in treatments with lower levels of IBA

and treatments with fewer that the maximum of three applications of IBA during

their study. There also appears to be evidence that auxin concentrations above the

100 ppm treatment begin to negatively impact graft union success and plant vigor.

Single applications of 150 ppm IBA and two applications of 150 ppm IBA had a

lower percentage of successful graft unions than the same numbers of applications

of 100 ppm IBA.

Köse and Güleryüz (2006) showed that cytokinin applications can influence

the success of graft union formation. In Vitis vinifera, application of cytokinins to

the grafting site was shown to improve the number of successful grafts compared

to a control, yielding a 100% success rate under a 250 mg/l kinetin application.

Auxins improved rooting of grafted cuttings, but were detrimental to grafting

success, reducing the success rate to 0% under many auxin treatments (Köse &

Güleryüz, 2006).

Incompatibility

Some rootstock-scion pairings do not form graft unions, while others will

successfully form a union but exhibit poor growth and eventually die. In these

cases, the rootstock and scion are said to exhibit incompatibility (Andrews &

Marquez, 1993). The likelihood of having an incompatible rootstock-scion

combination increases as the rootstock and scion become more distantly related to

each other. Intra-specific grafts tend to be more successful than interspecific

grafts, and intra-generic grafts tend to be more successful than intergeneric grafts

(Hartmann et al., 2010). Estrada-Luna et al. (2002) reported such differences in

cactus. Interspecific in vitro micrografts of Opuntia ficus-indica on O. cochinera

9 9

and O. leucotricha showed 36% to 43% less growth than conspecific micrografts,

which was thought to be due to reduced physiological compatibility. Lohar &

VandenBosch (2005) demonstrated that intergeneric grafts between Lotus

japonicas and Medicago truncatula formed one third the number of successful

graft unions compared to self-grafts of L. japonicas.

Incompatibility is caused by a physiological intolerance between different

cells, but the mechanism by which this intolerance occurs is not clear. There are

three main mechanisms (barring the presence of pathogens) proposed to explain

incompatibility: 1) toxicity between the rootstock and scion; 2) phenolics that

inhibit proper union formation; and 3) hormonal abnormalities.

Incompatibility between pear (Pyrus communis) cultivars on quince

rootstocks has been attributed to rootstock toxicity (Gur, Samish, & Lifshitz,

1968). Quince (Cydonia oblonga) produces prunasin, which breaks down to

produce cyanide, an inhibitor of cellular respiration that can lead to the death of

phloem cells. Among pear cultivars, ‘Bartlett’ is incompatible with quince, while

‘Old Home’ can be successfully grafted onto quince rootstocks. This difference in

compatibility is the result of a physiological difference; ‘Old Home’ produces an

enzyme that detoxifies prunasin, while ‘Bartlett’ lacks the enzyme and dies from

cyanide poisoning. However, cyanide-containing compounds are not present in

many species of plants, so this cannot be the sole reason for graft incompatibility.

The presence of phenolic compounds has also been linked to graft

incompatibility (Evans & Rassmussen, 1972). Phenolics are produced in response

to stress and wounding, both of which occur during grafting. Additionally, they

are involved in lignification, which is an important step in the formation of a

successful graft union.

10 10

A growing body of evidence also supports the hormonal abnormality

hypothesis. Auxin in particular is implicated in delayed incompatibility (Aloni et

al., 2008).

Rootstock-Scion Interaction

Once the graft union has successfully formed, there is a complete vascular

connection between the rootstock and scion. This allows for the transfer of

materials between the rootstock and scion via the xylem and phloem, and also via

the parenchyma cells surrounding the vasculature. It is clear that rootstock and

scion tissues communicate (Hartmann et al., 2010), as demonstrated by so-called

“rootstock effects.” Certain rootstocks are associated with increased vigor or

dwarfing when combined with certain scions, which is thought to be hormonally

mediated. Recent research has demonstrated a variety of molecules are exchanged

between rootstock and scion, including genetic material (Fuentes, Stegemann,

Golczyk, Karcher, & Bock, 2014).

Hormone signaling. Hormones are exchanged between rootstocks and

scions of grafted plants. Auxins from the shoot apices of the scion travel down to

the roots of the rootstock, cytokinins from the rootstock travel upward into the

scion, and both have effects on growth and morphology in their respective

destinations. This “hormone message concept” has been extensively tested and

now provides a working model for the examination of the many processes that

result from grafting (Aloni et al., 2010). Bangerth (1994) found that cytokinin and

auxin levels in plants are regulated through a feedback loop. As levels of IAA

(indole acetic acid) decrease, cytokinin production in the roots is stimulated. Once

cytokinin levels in the xylem rise to sufficient levels, IAA production and

translocation are stimulated. The rise in IAA then causes a reduction in cytokinin

11 11

levels in the xylem. Through this constant ebb and flow of antagonistic hormones,

an un-grafted plant maintains a balanced cytokine/auxin ratio (Sorce, Massai,

Picciarelli, & Lorenzi, 2002). However, in a grafted plant the hormone levels may

be unbalanced, which could explain the resulting “rootstock effects” seen in

grafted trees. Rootstocks that confer vigor would produce higher levels of

cytokinin relative to the scion’s auxin levels (which would also reduce overall

auxin levels), resulting in greater shoot growth and increased branching. In Van

Hooijdonk, Woolley, Warrington, and Tustin (2010), rootstocks of differing vigor

were demonstrated to affect the architecture of commercial scions of ‘Royal Gala’

apple. The dwarfing rootstock, ‘M.9’, had a similar effect on branching and shoot

growth as an application of auxin transport inhibitor. Applications of a cytokinin

(BAP) reversed the dwarfing effects of the ‘M.9’ rootstock. This effect suggests

that dwarfing rootstocks produce low concentrations of cytokinin relative to auxin

levels in the scion, perhaps due to inhibition of auxin transport. Inhibition of auxin

transport has been shown to prevent symptoms of graft incompatibility in

normally incompatible watermelon/pumpkin combinations (Aloni et al., 2008).

Genetic exchange. That grafted plants might be able to hybridize without

sexual reproduction is a long held idea, perhaps first articulated by Darwin (1868)

in his The Variation of Animals and Plants Under Domestication. Other notable

proponents of the “hybridization by grafting” theory include Hans Winkler and

Ivan Michurin. Winkler conducted work on Solanum spp. and demonstrated that

polyploid plants could be produced from grafting (Zhou & Liu, 2015). Michurin

invented the process of “mentor grafting,” in which seedlings are grafted to mature

plants (Liu, Wang, & Li, 2011; Zhou & Liu, 2015). He demonstrated that

seedlings grafted in this method developed characteristics similar to the mentor

12 12

specimen. Small pieces of genetic material (mRNA, nucleic acids) have been

observed passing between rootstocks and scions (Gahan, 2013), so it is

conceivable that more substantial translocations of genes or groups of genes could

happen. However, conclusive evidence of this phenomenon did not exist until

2014, when Fuentes et al. showed that two grafted plants of different species can

produce material that represents a new allopolyploid species, with a genome that

has the same number of chromosomes as the sum of its two parent species. In

Fuentes et al.’s study, two species of tobacco (Niccotiana glauca and N. tabacum)

were grafted together. Each plant had been transformed to express resistance to a

different antibiotic. Once a successful graft union had formed, pieces of the graft

junction were placed on a medium that contained both antibiotics. The pieces of

graft junction that successfully grew on the test medium were grown into plants

and allowed to flower. The plants derived from the dual resistant plant material

exhibited many traits that were intermediate between the two parent species. Leaf

color and shape, flower color, flower shape, and flower size all appeared as a

blend between the character states of N. glauca and N. tabacum. Plant height and

chromosome count were larger than either parent species, which is often seen in

polyploidy. However, most polyploid specimens show a straightforward doubling

of the genome and therefore contain twice the number of chromosomes as the

parent species. Since the new tobacco species (dubbed N. tabauca) is the result of

combining two different species, its chromosome count reflects that fact. N.

glauca has 24 chromosomes, N. tabacum has 48 chromosomes, and the N. tabauca

contains 72 chromosomes. This finding shows that the new species contains the

full nuclear genomes of the two parent species, rather than just the sections of

DNA that confer antibiotic resistance.

13 13

Practical Implications

Grafting is an ancient technique that remains valuable in modern

agriculture. Recent evidence has furthered our understanding of how graft unions

form (successfully and otherwise) and how rootstocks and scions communicate to

influence each other during and after graft union formation. Very recent work has

confirmed the long-held belief that grafting can produce hybrids without sexual

reproduction. Understanding the physiological underpinnings of grafting will

allow for greater manipulation of grafted plants in the field. Because much of

rootstock/scion interactions are mediated by plant hormones that are commercially

available, manipulations of grafting physiology should be relatively feasible to

implement on a large scale. Manipulation of grafting physiology has already been

conducted in apple with an eye toward commercial production (Cummins, 1997)

and other examples are sure to appear as academic findings make their way out

into industry.

Cactus Pear (Opuntia ficus-indica) Origin, Cultivation, and Uses

Opuntia ficus-indica originated in central Mexico (Casas & Barbera, 2002)

and has since spread throughout the globe and is found on all continents except

Antarctica (Inglese, Basile, & Schirra, 2002). The plant species has been used by

humans for millennia (Casas & Barbera, 2002). The succulent pads (cladodes) are

eaten as a vegetable when young and tender (Colunga Garcia-Marin, 1984), while

older cladodes have shown promise as supplemental fiber in baked goods (Ayadi,

Abdelmaksoud, Ennouri, & Attia, 2009). Cladodes of any age can be used as

forage for livestock, and can help livestock survive droughts by providing much-

needed moisture (Mondragón-Jacobo & Pérez-González, 2001). O. ficus-indica

has also been used to treat a wide range of ailments, including inflammation

14 14

(Casas & Barbera, 2002), and diabetes (Frati, Jiménez, & Ariza, 1990). The

mucilage produced from cut cladodes can be used as an adhesive and the dried

skeletons of the plants can be used as fire wood (Bravo-Hollis, 1978; Colunga

Garcia-Marin, 1984). The fruit is eaten fresh or processed into jellies, candies, and

beverages (Lee, Pyo, Ahn, & Kim, 2005; Sawaya, Khatchadourian, Safi, & Al-

Mouhammad, 1983). The total world crop production was estimated at 300,000

tons of fruit. Mexico is the largest producer, with an estimated production of

200,000 tons of fruit on an area of 50,000 ha (Flores & Gallegos, 1994). Italy,

Spain, Egypt, Morocco, and Israel are other important producers, with minor

production in South America, South Africa, Southwestern Asia, the U.S., and

Australia. Fruits are typically sold locally, although Mexico, Italy, and Chile also

provide fruit for export (Tous & Ferguson, 1996). Within the United States, a

majority of cactus pear is produced in California. Fruit production can be a very

profitable business for growers; for example, 450 acres in Monterey County

grossed around $2 million in 1998 (Crop Profile for Cactus Pear in California,

2000). Recent studies of the fruit from cacti grown on poor quality soils (e.g., high

in salinity and selenium) show that the fruit accumulates selenium in the form of

selenoamino acids, which have been suggested to possess anti-carcinogenic

properties (Banuelos et al., 2012).

15

CHAPTER 1: ESTABLISHMENT OF ASEPTIC CULTURES AND SHOOT INDUCTION IN THREE CULTIVARS OF

SALT-TOLERANT CACTUS PEAR (OPUNTIA FICUS-INDICA)

Introduction

Opuntia ficus-indica has a well-documented history of successful in vitro

propagation. However, salt-tolerant cultivars of cactus have yet to be evaluated for

in vitro propagation potential. The “Seleno” series of salt-tolerant cactus are a

potential new crop for salt-affected agricultural lands (Banuelos & Lin, 2010). In

vitro production of these unique cultivars will allow for large-scale production of

plants for field plantings and can facilitate other in vitro techniques, like

micrografting or physiological studies. The initial stages of such production

include establishment of sterile culture and shoot induction.

Materials and Methods

Plant Material

Mature overwintered cladodes and young tender cladodes of an unnamed

Opuntia ficus-indica cultivar were collected from a private residence in Del Rey,

CA, located 12 miles from the USDA-ARS in Parlier, CA for use in initial

sterilization experiments. The remaining experiments used young tender cladodes

that were collected from USDA-ARS in Parlier during the period of active growth

or produced in the greenhouses at the California State University, Fresno

Horticulture Unit from the Parlier material.

Experiment 1-Harsh Sterilization

The first relatively harsh sterilization method was as follows: whole

cladodes were immersed for 10 min in 20% bleach, 30 min in benzalkonium

16 16

chloride (1.1856%) Lysol®, and were rinsed four times with distilled water

(Mohamed-Yasseen, Barringer, Splittstoesser, & Schnell, 1995). After surface

sterilization, cladodes were cut into approximately 2cm x 2cm squares including at

least one areole, with one square per magenta box. Nineteen magenta boxes were

used, with 50 ml of medium in each box. The medium (El Finti, El Boullani, El

Ayadi, Ait Aabd, & El Mousadik, 2012) was MS (Murashige & Skoog, 1962)

supplemented with 50 g/l sucrose, 50mg/l monosodium phosphate, 40 mg/l

adenine sulfate, 3g/l agar, 5mg/l BA, adjusted to pH 5.7 and autoclaved at 121º C

for 30 min after pouring into culture vessels. Plants were maintained in a growth

room under a 16-h day. Light intensity was 500 µmol m-2 sec-1 PPFD provided by

fluorescent lamps and temperature was maintained at 25º C ± 2º C.

Contamination was assessed and recorded for 2 weeks.

Experiment 2-Harsh + Brush Sterilization

Harsh + Brush Sterilization was a modified version of Harsh Sterilization.

All steps remain the same, but whole mature cladodes were brushed with a 9:1

ratio of 90% ethanol to Lysol mixture for 5 min with a toothbrush and rinsed

under tap water before proceeding to the bleaching step. After surface sterilization,

the cladodes were cut into approximately 3 x 3 cm squares that included at least

one areole. Forty-eight culture jars with 20 ml of medium per jar were used. The

medium is of the composition laid out by El Finti et al. (2012). The medium was

adjusted to pH 5.7 and autoclaved at 121º C for 30 min after pouring into culture

vessels. Each culture jar received one square of cladode. Contamination was

assessed and recorded each week for 4 weeks.

17 17

Experiment 3-Harsh + Swirl Sterilization

Harsh + Swirl Sterilization was a modified version of Harsh + Brush

Sterilization. Instead of brushing the cladodes with a toothbrush for 5 min, the

whole cladodes were gently swirled in the 9:1 mixture of 90% ethanol to Lysol for

30 s. Tender young cladodes of the unnamed cultivar that were 1-4 inches in

length were used. After surface sterilization, the cladodes were cut into

approximately 3 x 3 cm squares with at least 1 areole. Twenty-five magenta boxes

with 40 ml of medium per box were used as culture vessels. The shoot induction

medium was made using the formulation in El Finti et al. (2012). The medium was

adjusted to pH 5.7 and autoclaved at 121º C for 30 min after pouring into culture

vessels. One piece of cladode was placed in each magenta box. Contamination

was observed and recorded each week for 4 weeks.

Experiment 4- Different BA Concentrations on El Finti et al. (2012) Medium

A second trial of Harsh + Swirl Sterilization was conducted using patented

O. ficus-indica cultivars. Tender young cladodes from cultivars Seleno-Red (252),

Seleno-Purple(248), and Seleno-Orange(255) were used. After surface

sterilization, the cladodes were cut into approximately 3 x 3 cm squares with at

least one areole. Four pieces of cladode were placed in each culture vessel (Figure

3), and 158 magenta boxes with approximately 40 ml of medium per box were

used. Fifty boxes each were allotted to cultivar 252 and 255, with 58 boxes

allotted to cultivar 248. MS medium with 50g/l sucrose, 50mg/l monosodium

phosphate, 40 mg/l adenine sulfate, and 3 g/l agar, adjusted to pH 5.7 (El Finti et

al., 2012) was used with 4 different BA concentrations: 0 mg/l (control), 5mg/l

(low), 7.5 mg/l (medium), and 10 mg/l (high). Ten magenta boxes of each of the

18 18

Figure 3. Explants, shoot initiation, growth, and proliferation

19 19

control, medium and high levels of BA were allotted to each cultivar. Twenty

boxes each of low BA were allotted to cultivars 255 and 252, while 28 boxes of

low were allotted to cultivar 248. Explants were placed on shelves under lights in

a growth room. A 16-h day was maintained, with a light intensity of 500 µmol m-2

sec-1 PPFD provided by fluorescent lamps and the temperature was maintained at

25º C ± 2º C. Contamination, health of explants, and shoot initiation were

assessed and recorded each week for 7 weeks.

Gentle Sterilization

Gentle Sterilization was as follows: whole tender young cladodes were

washed in soapy water for 5 min, then swirled in 70% ethanol for 30 s, then placed

in 10% bleach and Tween 20 for 10 min and then rinsed 3 times with sterile DI

water (Kitchen Culture Technologies Inc., 2010). This method was used 3 times as

part of alternative media formulation experiments.

Experiment 5- medium with GA3 vs medium without GA3. Surface

sterilization followed the Gentle Sterilization protocol. After surface sterilization,

young tender cladodes were cut into approximately 3 x 3 cm squares with at least

one areole. Two media, one containing gibberellic acid (Llamoca-Zarate, Aguiar,

Landsmann, & Campos, 1999) and one without gibberellic acid (El Finti et al.,

2012) were prepared for use on greenhouse-grown cladodes of cultivars 255 and

252. Specifically, the Llamoca-Zarate et al. medium was MS medium with 50 g/l

sucrose, solidified with 8 g/1 agar and supplemented with 2.22 µM BA and 1.44

µM gibberellic acid (GA3) (Llamoca-Zarate et al., 1999). The medium without

GA3 was the El Finti et al. medium. All media were adjusted to pH 5.7 and

autoclaved at 121º C for 30 min after pouring into culture vessels. Two pieces of

cladode were placed in each culture vessel. Three magenta boxes of each media

20 20

were prepared for each cultivar for a total of six boxes. Contamination and shoot

induction were observed and recorded each week for 4 weeks.

Experiment 6-medium with IBA vs medium without IBA. Surface

sterilization followed Gentle Sterilization protocol. After surface sterilization,

cladodes were cut into approximately 3 x 3 cm squares with at least one areole.

Two other media were prepared for field-grown cladodes of cultivars 248, 252, and

255. Media with indole-3-butyric acid (IBA) (Juárez & Passera, 2002) and without

IBA (Estrada-Luna, 1988) were compared. Specifically, the medium from Juárez

and Passera contained MS medium with 2.25 mg/l BA, 2.03 mg/l IBA, 30 g/l

sucrose and 8g/l agar. The Estrada-Luna (1988) medium contained MS medium

supplemented with 50 g/l sucrose, 7 g/l agar, and 2.5 g/l agar. All media were

adjusted to pH 5.7 and autoclaved at 121º C for 30 min after pouring into culture

vessels. Two to three squares were placed in each culture vessel and 30 magenta

boxes of each medium were prepared. Ten boxes of each medium were allotted to

cultivar 255, five boxes of each medium were allotted to cultivar 248, and five

boxes for each medium were allotted to cultivar 252. Contamination and shoot

induction were observed and recorded each week for 4 weeks.

Experiment 7-greenhouse-grown explants on medium without IBA.

Fifteen magenta boxes of the Estrada-Luna (1988) medium were prepared for

greenhouse-grown cladodes of cultivars 255 and 248. Ten boxes with 50 ml of

medium per box were allotted to cultivar 255 and five boxes were allotted to cultivar

248. Surface sterilization followed gentle sterilization, after which the cladodes were

cut into approximately 3 x 3 cm squares with at least one areole. Contamination and

shoot induction were observed and recorded each week for 4 weeks. Table 1

summarizes all experimental conditions.

21

Table 1

Summary of Experimental Conditions

Experiment

Sterilization

Protocol

Media

Cultivars Used

Number of Replicates

PGR Concentration

Age of

Cladodes

Origin of

Cladodes

1 Harsh El Finti et al. (2012) unnamed 19 5mg/l BA Mature Field

2 Harsh + Brush El Finti et al. (2012) unnamed 25 5mg/l BA Mature Field

3 Harsh + Swirl El Finti et al. (2012) unnamed 48 5mg/l BA Young Field

4

Harsh + Swirl

El Finti et al. (2012)

248

252

255

10 per cultivar 0mg/l BA Young Field

4

Harsh + Swirl

Modified El Finti et al.

(2012)

248

252

255

28 for 248

20 for 252

20 for 255

5mg/l BA Young Field

4

Harsh + Swirl Modified El Finti et al.

(2012)

248

252

255

10 per cultivar 7.5mg/l BA Young Field

4

Harsh + Swirl Modified El Finti et al.

(2012)

248

252

255

10 per cultivar 10mg/l BA Young Field

5

Gentle El Finti et al. (2012) 252

255

3 per cultivar 5mg/l BA Young Greenhouse

5

Gentle Llamoca-Zarate et al.

(1999)

252

255

3 per cultivar 0.5mg/l BA, 0.5 mg/l

GA3

Young Greenhouse

6

Gentle Juarez & Passera (2002) 248

252

255

5 for 248

5 for 252

10 for 255

2.25mg/l BA, 2.03 mg/l

IBA

Young Field

6

Gentle

Estrada-Luna (1988)

248

252

255

5 for 248

5 for 252

10 for 255

2.5 mg/l BA Young Field

7

Gentle Estrada-Luna (1988) 248

255

5 for 248

10 for 255

2.5 mg/l BA Young Greenhouse

22 22

Statistical Analysis

Statistical analysis was performed with IBM SPSS Version 24 software.

Contamination and shoot initiation data were recorded in a present/absent format

with present coded as ‘1’ and absent coded as ‘0’. The data were not normally

distributed, so Kruskal-Wallis and Mann-Whitney U tests were used to detect

differences between treatments.

Results

Contamination

For Harsh Sterilization, 18 boxes were contaminated (94.7%

contamination) after 2 weeks. Some explants produced shoots in contaminated

culture vessels. Fungal pathogens were the primary contaminants.

After 4 weeks, the 42 jars that had been treated with Harsh + Brush

Sterilization were contaminated (87.5% contamination). Fungal contamination

appeared in three locations within a culture vessel: 1) isolated spots on the

medium away from any explants, 2) on the edges or sides of explants with contact

to the medium, and 3) on or around the areoles of the explants without contact

with the medium. Contamination originating in the areole was noticed starting at 2

weeks, and it was the main source of new contamination at 4 weeks. At 4 weeks,

71.4% of jars that with newly documented contamination had fungal growth from

the areole (Figure 4). There was no significant difference in contamination rate

between Harsh Sterilization and Harsh + Brush Sterilization (Table 2).

Contamination for Harsh + Swirl Sterilization was 10.1%. Harsh + Swirl

Sterilization was observed to be significantly different from Harsh Sterilization

(p<0.001) and Harsh + Brush Sterilization (p<0.001). A significant difference

23 23

Table 2

Contamination and Shoot Induction Rates by Sterilization Method, Media, Origin

and Age of Cladodes Used for Initiation of Sterile Cultures. Shoot Initiation Rates

Calculated for Clean Explants of Patented Cultivars Only Sterilization

Method

Media

Origin

Age

%

Contamination

% Shoot Initiation

from Clean

Explants

Harsh El Finti et al.

(2012)

Field Mature 94.7 -

Harsh +

Brush

El Finti et al.

(2012)

Field Mature 87.5 -

Harsh +

Swirl

El Finti et al.

(2012)

Field Young 10.1 23

Gentle Estrada-Luna

(1988), Juarez &

Passera (2002)

Field Young 100 -

Gentle Estrada-Luna

(1988)

Greenhouse Young 20 77.8

Figure 4. Examples of fungal contamination arising on and around the areole

24 24

in contamination level was detected between mature and young field-grown

cladodes (p < 0.001). Overall, there was no significant difference in

contamination level between the patented cultivars. Shoot induction for

successfully sterilized boxes was observed to differ significantly (p<0.001)

between Harsh + Swirl Sterilization (23%) and Gentle Sterilization (77.8%). A

significant difference in contamination rate (p<0.001) was observed between field-

grown material and greenhouse-grown material treated with Gentle Sterilization.

There was no significant difference in the rate of shoot induction between the

medium with GA3 (Llamoca-Zarate et al., 1999) and the medium without GA3 (El

Finti et al., 2012). No meaningful comparison between the medium with IBA

(Juárez & Passera, 2002) and the medium without IBA (Estrada-Luna, 1988)

could be made because all culture vessels were contaminated, but a single shoot of

255 was produced on the medium without IBA before the culture vessel

succumbed to contamination.

In the trials using patented cultivars, two distinct morphologies of shoot

were produced: normal, elongated shoots that resembled cactus seedlings and

abnormal shoots that seemed to increase more in diameter than in length as they

grew. Some shoot multiplication occurred after the first shoots were produced.

Axillary buds on the initiated shoots were activated and produced additional

smaller side shoots. Abnormal explants from cultivar 248 were observed in

abnormal shoots on El Finti et al. (2012) medium, but not on Estrada-Luna (1988)

medium.

Different BA Concentrations in El Finti et al. (2012) Medium

Total Shoot induction for cultivars 248, 252, and 255 on El Finti et al.

(2012) medium over all BA levels was 29.3%, 26 %, and 2%, respectively. There

25 25

was no significant difference in shoot induction between cultivar 248 and cultivar

252. Cultivar 255 did differ significantly from the other two cultivars (p<0.001).

There was no significant difference in shoot induction between any of the cultivars

at the control and high BA levels. A significant difference between cultivar 255

and both the 248 and 252 cultivars was detected for the low and medium BA

concentrations (p = 0.001 &0.049, p = 0.009 & 0.029, respectively). A significant

difference was detected between cultivars 248 and 255 at the low BA

concentration (p = 0.038), but not at the medium BA level.

Discussion

Harsh sterilization and Harsh + Brush Sterilization did not provide a high

degree of surface sterilization. Harsh + Swirl Sterilization is almost identical to

Harsh Sterilization and Harsh + Brush Sterilization, yet Harsh + Swirl Sterilization

had a much lower rate of contamination. The important difference appears to be

the age of the cladodes at the time of sterilization. The mature cladodes from

Harsh Sterilization and Harsh + Brush Sterilization had been growing in the field

for at least 1 year before they were harvested, while the young tender cladodes

used in the Harsh + Swirl Sterilization protocol were less than 6 months old. The

young cladodes spent less time exposed to potential contaminants and probably

had a lower overall contaminant load. Additionally, morphological differences

between mature and young cladodes may have contributed to differences in

contamination rate. In mature cladodes, the areole is covered in densely-packed

hairs and the surrounding cuticle has visible pits that house the stomata scattered

across its surface. Both of these structures appear to harbor fungal pathogens

(Figure 4) and may protect them during surface sterilization (Garcıa-Saucedo,

Valdez-Morales, Valverde, Cruz-Hernandez, & Paredes-Lopez, 2005). These

26 26

structures are not well-developed on young cladodes and likely provide a less-

secure refuge for pathogens. Source location also appeared to play a role in

successful surface sterilization in Gentle Sterilization. Field grown plants were

universally contaminated, but 80% of greenhouse-grown cladodes were

successfully sterilized. This indicates that greenhouse-grown plants are inherently

cleaner than field-grown plants and so can be successfully sterilized with a more

gentle method.

BA concentration was an important factor in shoot induction. BA

concentrations that were too low failed to induce shooting, while BA

concentrations that were too high seemed to inhibit shoot induction. For cultivars

252 and 248, treatment with 5 mg/l BA resulted in a good rate of shoot induction,

but many of the explants produced were of abnormal morphology per culture

vessel (30.6±12.1 % and 49.5.5±15.6%, respectively). The Estrada-Luna (1988)

medium utilizes a BA level of 2.5 mg/l. This medium showed an even higher rate

of shoot induction than El Finti et al. (2012) medium and there was no incidence

of abnormal morphology in any of the induced shoots for cultivar 248. Cultivar

252 was not successfully tested on Estrada-Luna (1988) medium, but it has been

shown to respond to BA in a similar manner to cultivar 248. For cultivar 255, all

levels of BA tested using El Finti et al. medium appeared to be too high and

inhibited shoot induction. Shoots were induced on the Estrada-Luna (1988)

medium, but a very high incidence of abnormal shoots (89.3±2.1 %) was

observed. This suggests that cultivar 255 requires lower BA concentrations than

the other cultivars for successful shoot induction and to produce normal shoots.

The low rate of shoot induction on El Finti et al. medium does not agree with

findings in the literature (El Finti, 2012; Khalafalla, Abdellatef, Mohameed-

Ahmed, & Osman 2007). The contrary findings may be due to cultivar differences,

27 27

differences in harvest technique, or differences in cultural practices for source

plants between studies. Shoot induction on the Estrada-Luna (1988) medium

supported previous findings (Estrada-Luna, 1988). Abnormal shoots have been

observed in Mammilaria pectinifera, Pelecyphora aselliformis (Giusti et al.,

2002), and Cereus peruvianus (Maria de Fátima & Prioli, 1996) explants placed

on shoot induction medium, but the rate of abnormal shoot formation was very

low.

The findings of this study are instructive in the construction of an in vitro

production method for salt-tolerant cactus pear. Greenhouse-grown cladodes

should be used as the source material and sterilized with Gentle Sterilization.

Shoots could be induced in cultivars 252 and 248 on Estrada-Luna et al. (1988)

medium. The exact concentration of BA needed to induce mostly normal shoots in

cultivar 255 is as yet unknown, but will be less than 2.5 mg/l.

28

CHAPTER 2: IN VITRO SHOOT PROLIFERATION, ROOTING AND MICROGRAFTING OF THREE CULTIVARS OF SALT-

TOLERANT CACTUS PEAR (OPUNTI FICUS-INDICA)

Introduction

In vitro production of cactus pear (Opuntia ficus-indica) has been examined

for its potential in large-scale production of plants for field planting and further in

vitro studies (El Finti et al., 2012) This study was specifically aimed at developing

a cultivar-specific production system for three unique cultivars developed and

patented by the USDA and California State University, Fresno. The “Seleno”

series of prickly pear cactus (Orange, Red, and Green, with accession numbers

255, 252, and 248, respectively) were selected for salt-tolerance and have the

additional characteristic of accumulating selenium-based antioxidants in their fruit

when grown on soils contaminated with selenium. They can produce a value

added crop on saline soils and tolerate minimal irrigation with poor-quality water.

These qualities make them an attractive option for farmers in the western San

Joaquin Valley, who are facing increasing shortages of surface water for irrigation

and/or leaching of naturally-occurring salts from the soil (Diffenbaugh et al.,

2014). However, there are differences between cultivars (Banuelos et al., 2012),

with higher salt-tolerance in cultivar 255, and desired fruiting characteristics in

cultivars 252 and 248. One way to quickly combine desired characteristics is to

graft. Preliminary attempts at in situ grafting suggested that desiccation and

contamination are major obstacles to grafting success. To circumvent these issues

and produce large numbers of grafted plants in a short time, in vitro techniques

were employed. The previous section described the development of protocols for

establishment of sterile cultures, so this chapter will delve deeper into shoot

proliferation, root induction and in vitro grafting.

29 29

Materials and Methods

Shoot Proliferation

In vitro-produced shoots from the three cultivars were placed on one of two

shoot induction media. Each culture vessel received one shoot. Shoots that were

approximately 3 cm long were cut in half longitudinally and placed on the media.

Cultivars 248 (n= 3) and 255 (n=4) were placed on MS medium (Murashige &

Skoog, 1962) supplemented with 50 g/l sucrose, 7 g/l agar, and 2.5 g/l agar

(Estrada-Luna, 1988). Cultivars 248 (n= 3) and 252 (n=4) were placed on MS

medium supplemented with 50 g/L sucrose, 50mg/L monosodium phosphate, 40

mg/L adenine sulfate, 3g/L agar, 5mg/l BA. All media were adjusted to pH 5.7 and

autoclaved at 121º C for 30 min after pouring into culture vessels. Total number of

shoots produced, number of normal shoots produced, and number of abnormal

shoots produced was observed after 18 weeks.

Root Induction

In vitro-produced shoots from cultivar 255 were used for root induction.

Five explants of normal phenotype and six of abnormal phenotype shoots were

placed on a rooting medium consisting of ½ strength MS medium supplemented

with 30 g/l sucrose, 3g/l agar, and 0.5 mg/l IBA (El Finti et al., 2012). The

medium was adjusted to pH 5.7 and autoclaved at 121º C for 30 min after pouring

into culture vessels. Each culture vessel received one explant. The number of

shoots produced by each explant was recorded after 13 weeks.

In vitro Grafting

In vitro-produced shoots of cultivars 255 and 252 were selected. Six

representatives each of normal and abnormal phenotype shoots from cultivar 255

were used as rootstock material. All scion material was normal phenotype shoots

30 30

from cultivar 252. A horizontal graft was used to join the rootstock and scion. The

medium used was of the same composition as that from the root induction

experiment (El Finti et al., 2012) and was adjusted to pH 5.7 and autoclaved at

121º C for 30 min after pouring into culture vessels. Each culture vessel received

one grafted explant and was placed in specific lighting conditions.

Statistical Analysis

Statistical Analysis was performed with IBM SPSS version 24 software.

ANOVA and LSD means separation were performed on shoot proliferation data

and an Independent Samples T-test was used for the rooting data. The Mann-

Whitney U test was performed on the in vitro grafting data.

Results

No significant difference in total shoot production was observed between

treatments. No significant difference in number of normal or abnormal shoots was

observed between the cultivars on El Finti et al. (2012) medium. Significant

differences were observed in number of normal shoots, number of abnormal

shoots, and percent abnormal shoots between cultivars 248 and 255 when on

Estrada-Luna (1988) medium (p =0.028 ). Those same variables were found to be

significantly different between the cultivars on El Finti et al. medium and cultivars

on Estrada-Luna medium (p =0.004 ) (Table 3). A significant difference in roots

produced was observed between normal and abnormal shoots of cultivar 255 (p

<0.001) (Table 4).

All in vitro micrografts failed. Distribution of failure type was not

significantly different between rootstock phenotype. 50% of grafts failed due to

contamination, 25% failed due to death of the rootstock material, and 25% failed

due to death of the scion material (Table 5).

31 31

Table 3

Shoot Proliferation and Incidence of Normal and Abnormal Phenotypes

Cultivar

Medium

Mean

Total Shoots

Mean

Normal

Mean

Abnormal

Mean

% Abnormal

248 El Finti

et al.

(2012)

12.3 ± 3.9 a 6.3 ± 3 ab 6 ± 2 a 49.5 ±15.6 a

252 El Finti

et al.

(2012)

16 ± 2.3 a 11 ± 2.7 a 5 ± 2.3 a 30.6 ± 12.1 a

248 Estrada-

Luna

(1988)

22 ± 4.6 a 22 ± 5 b 0 ± 0 b 0 ± 0 b

255 Estrada-

Luna

(1988)

25.5 ± 4.9 a 2.8 ± 0.8 c 22.8 ± 9 c 89.4 ± 2.1 c

Table 4

Root Production by Phenotype Phenotype Mean Roots Produced

Normal 21.2 ± 1.5 a

Abnormal 1.0 ± 0.7 b

Table 5

Graft Failure by Type Failure Type % of Total Grafts

Contamination 50

Rootstock Death 25

Scion Death 25

Discussion

It is clear that the Estrada-Luna (1988) medium is better medium for use in

cultivar 248 and probably in 252 as well (cultivar 255 responds to different BA

concentration in a fashion nearly identical to that of cultivar 248). While there was

no difference in total shoots produced, the Estrada-Luna medium does not cause

any of the shoots produced to display the abnormal phenotype and can almost

32 32

double the number of normal shoots produced compared to the El Finti et al.

(2012) medium. Cultivar 255 has previously been shown to respond poorly to El

Finti et al. medium and had a high incidence of abnormal shoots on Estrada-Luna

medium. This observation suggests that a lower concentration of BA might be

necessary to eliminate production of abnormal shoots. If so, it should be possible

to replicate the success of cultivar 248 in cultivar 255 and produce 100% normal

shoots. Normal shoots are preferred to abnormal shoots because they are more

vigorous in vitro. Normal shoots of cultivar 255 produced more roots than

abnormal shoots by a factor of almost 20. Unfortunately, it was impossible to

determine if the differences between normal and abnormal shoots extended to in

vitro grafting. All of the attempted grafts failed. Contamination was the cause of

failure for half of the grafts, while death of either rootstock or scion material

doomed the other half of the grafts. The contamination was most likely introduced

during grafting due to the multiple transfers from separate vessels into a shared

vessel. The low success rate of in vitro grafts reported here is contrary to what is

found in the literature. Previous research demonstrated a grafting success rate of

90% in horizontal micrografts (Estrada-Luna et al., 2002). The discrepancy in

success rate could be due to cultivar difference between studies or because

abnormal explants were included. The results of this study provide information

that is important for the design of a successful in vitro production system for

cultivars 248, 252, and 255. Abnormalities in shoot phenotype are largely the

product of the shoot induction medium and can be eliminated by lowering the

level of BA in the medium. In vitro grafting should utilize only normal phenotype

shoots should be closely monitored for contamination and death of rootstock or

scion material in the future.

33

CHAPTER 3: IN SITU GRAFTING OF SALT-TOLERANT CACTUS PEAR (OPUNTIA FICUS-INDICA)

Introduction

The “Seleno” series of Opuntia ficus-indica (which includes cultivars 248

(Seleno-Purple), 252 (Seleno-Red), and 255(Seleno-Orange)), was selected for

salt-tolerance and has the added benefit of producing nutraceutical fruit on

selenium-contaminated soils. However, all desired characteristics do not occur on

the same plant, so grafting is necessary.

Cacti are frequently grafted, but Opuntia-on-Opuntia grafts are not

commonly practiced. These types of grafts have a reputation for being difficult to

perform due to desiccation and contamination (Maldonado & Zapien-Barragan,

1977). Despite these problems, two methods have been developed specifically for

interspecific grafting of Opuntia (Huffmann, 2003; Serimian, personal

communication, May, 6, 2016). However, they have not been attempted in the

“Seleno” series. These two techniques, along with a new technique design

specifically for this experiment, will be evaluated for their effectiveness in

248/255 and 252/255 grafts for the purpose of developing an in situ production

system.

Materials and Methods

Plant Material

Plant material was collected from USDA-ARS in Parlier California. Large

mature cladodes were collected during summer. Cultivar 255 served as the

rootstock, with cultivars 252 and 248 serving as scions.

34 34

Parallel Wedge Graft

Rootstock cladodes were cured indoors at approximately 25º C for 2 weeks

prior to rooting. Rootstock cladodes were rooted in damp sand and then

transferred into 3.8 liter nursery pots filled with a well-drained potting medium.

Grafting was performed inside a closed room. Rootstock and scion

cladodes were surface sterilized with 70% ethanol before grafting. For the parallel

wedge graft, a wedge of tissue was cut from the apical portion of the rootstock

cladode. A corresponding wedge of tissue was cut from the scion material. Cutting

tools were sprayed between cuts with 70% ethanol and a flamed with a butane

torch. The cut edges of the scion wedge were secured in place against the cut

surfaces of the rootstock cladode using sterilized toothpicks. Care was taken to

ensure that a high degree of contact between rootstock and scion cut surfaces was

achieved. The graft wound was then sealed with a quick-curing silicone sealant

(Figure 5). After the silicone had cured and was no longer tacky to the touch,

grafted plants were moved to a greenhouse. Plants were watered once the potting

soil had dried completely. Five grafts were made of each scion/rootstock

combination (252/255 and 248/255). The condition of the graft union and overall

plant health were observed for 4 weeks.

Horizontal Graft

Unrooted cladodes were used for ease of handling. Grafting was performed

in a closed windowless room. Work surfaces were sprayed with 70% ethanol prior

to grafting. Both rootstock and scion cladodes were dipped in 2% bleach solution

and allowed to dry before any cuts were made. Guide lines were marked on scion

cladodes and used to determine where to cut on a corresponding rootstock cladode

to best match cladode width and thickness at the site of the graft wound and were

marked on the rootstock cladodes. Cladodes were cut horizontally in a straight line

35 35

Figure 5. Parallel wedge graft progression from graft initiation to graft

failure

36 36

across the widest face of the cladode. The cut was made in a single stroke using a

long sharp knife to get the cleanest cut possible. Between uses, the knife was

sprayed with 70% ethanol and flamed with a butane torch. The horizontal graft

used the apical piece of scion cladodes and the basal piece of each rootstock

cladode. After cutting, the cut surfaces of the rootstock and scion cladodes were

aligned to maximize physical contact. Hot melt glue for use on rubber, nylon, PVC

and metal (Ace Hardware) was used to secure the two cladode pieces together and

to seal the graft. Hot glue was dispensed from a dual heat glue gun set to “high”.

Hot glue separates from cactus cuticle if the cuticle is not heat-treated (Serimian,

personal communication, May, 6, 2016), so the heating element of the hot glue

gun was pressed against the cuticle of the cladodes in areas that would receive the

hot glue. Hot glue was not applied into the graft wound, but rather applied in a

continuous layer that extended between the rootstock and scion. Additional

support struts of glue were applied to maintain stability of the graft before

successful union formation. One to two support struts extended from the main area

of hot glue application up and down each side of the grafted cladode (Figure 6).

After the hot glue had cooled, the grafted cladodes were potted into nursery pots

filled with a well-drained potting medium. Five 252/255 grafts and six 248/255

grafts were made. One 248/255 graft utilized a much younger piece of scion

material than the other grafts. Graft union success or failure and overall cladode

health were observed for 3 weeks.

Perpendicular Wedge Graft

Grafting was performed in a closed windowless room. This protocol used

tender cladodes that still had vestigial leaves or had recently lost them. Scion

material was obtained from potted mother cladodes maintained in a greenhouse.

37 37

Scion material was grafted onto young rootstock cladodes that had emerged from

potted mother cladodes. Both rootstock and scion were sprayed with 70% ethanol

and allowed to dry before cutting. All cuts were made with a scalpel that was

sprayed with 70% ethanol and allowed to dry between grafts. A scion cladode was

cut from the mother cladode. The bottom was then trimmed into a tapered wedge

shape. The cuticle was removed to expose the photosynthetic chlorenchyma where

the trimmed section of the scion cladode was thickest (at the top of the trimmed

portion). In the rootstock, a vertical cut of the same length as the trimmed portion

of the cladode was made down the center of the rootstock cladode. The scion piece

was inserted into the vertical cut in the rootstock rotated 90º relative to the

orientation of the rootstock cladode, so that from above, the two cladodes would

appear to form a “+” shape (Figure 7). Scion material was secured in place with a

few drops of super glue at the top of both sides of the graft and an elastic band was

tied around the graft wound to maintain constant pressure. Grafted cladodes were

then moved to a greenhouse to heal. Graft health and success or failure was

observed and recorded for 2 weeks.

Statistical Analysis

Statistical Analysis was performed with IBM SPSS version 24 software.

Due to the non-parametric nature of success/failure experiments, data were

analyzed with the Kruskal-Wallis Test and Mann-Whitney U Test.

Results

Parallel Wedge Graft

After 1 week, the 252/255 grafts showed yellowing at cut edges, with little

or no yellowing on cut edges of 248/255 grafts. Necrosis was apparent in all grafts

38 38

after 2 weeks (Figure 5). Interestingly, three of the 248/255 grafts did not show

yellowing, despite the necrosis. Three weeks after grafting, all but three grafts

showed substantial necrosis and had begun to desiccate. Only one 252/255 graft

and two 248/255 grafts were still alive. After 4 weeks, all grafts had failed due to

necrosis and desiccation. A fungal pathogen was identified as the cause of the

widespread necrosis. This grafting method apparently provided two extremes of

suboptimal conditions. The sealed graft wound remained too humid and promoted

fungal growth, which caused necrosis. As the necrosis progressed past the bounds

of the silicone sealant, the plant tissue began to desiccate.

Horizontal Graft

After 1 week, one 248/255 graft had failed because the rootstock and scion

material separated. This problem continued into week 2, with a total of three failed

Figure 6. Completed horizontal grafts

39 39

248/255 grafts and one failed 252/255 graft. By week 3, all but a single 248/255

graft had failed and scion pieces were shriveled. The surviving graft successfully

formed a graft union and began to grow.

Perpendicular Wedge Graft

One graft of each scion/rootstock combination died after 2 weeks. All scion

pieces showed noticeable water loss as evidenced by their wrinkled appearance,

but those that had successfully formed a graft union eventually became turgid and

began to grow (Figure 7).

No significant difference was observed between cultivars with respect to

grafting success, so all graft types were analyzed as one group. There was a

significant association between grafting method and graft success (p = 0.001), but

no significant difference in graft success between Parallel Wedge graft and

Figure 7. Initial stage of perpendicular wedge graft and successful perpendicular

wedge graft

40 40

Horizontal graft. A significant difference in graft success was observed between

Perpendicular Wedge graft and Parallel Wedge graft (p = 0.007), and between

Perpendicular Wedge graft and Horizontal graft (p = 0.006).

Table 6

Success Rates for Three Grafting Techniques Grafting Method % Success

Parallel Wedge Graft 0.0 a

Horizontal Graft 9.09 a

Perpendicular Wedge Graft 66.67 b

Discussion

Both Parallel Wedge grafts and Horizontal grafts had displayed successful

grafting in other Opuntia species and cultivars (Huffmann, 2003; Serimian,

personal communication, May, 6, 2016). However, they do not appear to work

well on mature cladodes of the “Seleno” series cultivars. The only successful

Horizontal graft used much younger scion material than the other Horizontal

grafts, so perhaps Horizontal and Parallel Wedge grafts would work well on

younger material. However, they have drawbacks independent of graft success.

Both of these methods make it difficult to observe the graft union to monitor its

health and the sealants are not able to contract as the scion material shrinks prior

to graft union formation. The Perpendicular Wedge graft does not rely on sealants,

but rather on a snug fit between rootstock and scion pieces. This technique allows

for easy monitoring of the graft union. The lack of sealant material also allows for

good air circulation around the graft wound, which may be the reason why

Perpendicular Wedge grafts had no contamination issues. Overall, the

Perpendicular Wedge graft was superior to the other grafting techniques tested

with respect to the “Seleno” Series (Table 6). This grafting technique can now be

used as the basis for developing grafted cactus pear for field plantings.

41

CHAPTER 4: CONCLUSIONS

In Situ Grafting

Based on the results of these three studies, in situ grafting via Perpendicular

Wedge graft is the most immediately applicable technique for production of

grafted salt-tolerant cacti and is most likely to be useful for commercial

production. The Perpendicular Wedge graft is easy to perform, requires very little

in the way of equipment and supplies, and should translate well to field settings

(Figure 8). The graft site is easy to observe and graft success can be ascertained

within a week.

Figure 8. A production system for field-grafted plants

42 42

In Vitro Production and Grafting

In vitro techniques have yet to be optimized for successful large-scale

production of grafted salt-tolerant cacti. The Estrada-Luna (1988) medium (2.5

mg/l BA) would be used for the scion cultivars (248 and 252), but the rootstock

cultivar (255) needs lower levels of BA than were used in these studies to avoid

the production of abnormal shoots. Once that medium formulation is determined,

shoot production of all varieties can commence and in vitro grafting can be

conducted. In vitro grafting had three major problems in this study that impeded

successful graft union formation- death of rootstock, death of scion, and

contamination.

Rootstock death may be prevented by avoiding abnormal shoots. Avoiding

abnormal shoots should be simple because abnormal shoots were not produced in

scion material at lower levels of BA. Reduction in the rate of abnormal shoot

formation should occur for the rootstock material once an appropriate BA

concentration is determined.

Scion death may be prevented by selecting large healthy shoots for scion

material. The scion material is not in contact with the growth medium and cannot

receive nutrients from the rootstock until the vascular systems are connected, so

the grafted scion material needs to contain sufficient reserves of nutrients to

survive for the 30 days required to successfully form a graft union (Estrada-Luna

et al., 2002).

Contamination was either introduced during the grafting process or was

present-but-dormant on the parent material for the in vitro-produced shoots. If

contamination is not present after an abundantly cautious sterile technique is

followed, then the contamination was introduced during the grafting process.

Contamination that persists even after additional steps are taken to avoid external

43 43

sources of contamination indicates that dormant fungal spores may be present in

the apparently sterile cultures. This mode of contamination would be an extreme

version of the delayed-onset contamination that was observed in the first two

surface sterilization trials. Fungal spores may be present on the parent material and

lay dormant until disturbed by the grafting process. If this is determined to be the

case, in vitro–produced shoots would need to undergo further surface sterilization

and a further shoot proliferation step to ensure that contamination from the parent

material is no longer present before grafting. If cultures display contamination

after multiple rounds of surface sterilization, endogenous contamination is most

likely present. In vitro cultures would need to be screened for the pathogen and

infected explants would need to be disinfested or discarded (Leifert, Ritchie, &

Waites, 1991). Overall, the in vitro grafting system demonstrated in Estrada-

Luna (2002) does not work well for the “Seleno” series of Opuntia ficus-indica

cultivars, but can be adapted to meet their specific hormone requirements.

However, since an effective in situ grafting technique has been developed, an in

vitro production system is unnecessary until such time that the in situ production

system is rendered inefficient due to disease or unmet demand for grafted plants.

44

REFERENCES

Aloni, B., Karni, L., Deventurero, G., Levin, Z., Cohen, R., Katzir, N., Lotan-

Pompan, M., Edelstein, M., Akta, H., Turham, E., Joel, D.M., Horev, C., &

Kapulnik, Y. (2008). Physiological and biochemical changes at the rootstock-

scion interface in graft combinations between Cucurbita rootstocks and a

melon scion. The Journal of Horticultural Science & Biotechnology, 83(6),

777-783.

Aloni, B., Cohen, R., Karni, L., Aktas, H., & Edelstein, M. (2010). Hormonal

signaling in rootstock–scion interactions. Scientia Horticulturae, 127(2),

119-126.

Andrews, P. K., & Marquez, C. S. (1993). Graft incompatibility. Horticultural

Reviews, 15, 183-232.

Ayadi, M. A., Abdelmaksoud, W., Ennouri, M., & Attia, H. (2009). Cladodes

from Opuntia ficus indica as a source of dietary fiber: Effect on dough

characteristics and cake making. Industrial Crops and Products, 30(1), 40-

47.

Bangerth, F. (1994). Response of cytokinin concentration in the xylem exudate of

bean (Phaseolus vulgaris L.) plants to decapitation and auxin treatment, and

relationship to apical dominance. Planta, 194, 439-442.

Banuelos, G. S., & Lin, Z. Q. (2010). Cultivation of the Indian fig Opuntia in

selenium‐rich drainage sediments under field conditions. Soil Use and

Management, 26(2), 167-175.

Banuelos, G. S. et al. (2012). Biofortified, selenium enriched, fruit and cladode

from three Opuntia Cactus pear cultivars grown on agricultural drainage

sediment for use in nutraceutical foods. Food Chemistry, 135(1), 9-16.

Banuelos, G., Freeman, L., & Diener, J. (2013a). Opuntia plant named ‘Seleno-

Green’.U.S. Patent No. US PP24,053 P3. Washington, DC: U.S. Patent and

Trademark Office.

Banuelos, G., Freeman, L., & Diener, J. (2013b). Opuntia plant named ‘Seleno-

Orange’.U.S. Patent No. US PP24,039 P3. Washington, DC: U.S. Patent and

Trademark Office.

45 45

Banuelos, G., Freeman, L., & Diener, J. (2013c). Opuntia plant named ‘Seleno-

Purple’.U.S. Patent No. US PP24,072 P3. Washington, DC: U.S. Patent and

Trademark Office.

Banuelos, G., Freeman, L., & Diener, J. (2013d). Opuntia plant named ‘Seleno-

Red’.U.S. Patent No. US PP24,052 P3. Washington, DC: U.S. Patent and

Trademark Office.

Bravo-Hollis, H. (1978). Las cactáceas de México. Mexico City, Mexico:

Universidad Autónoma de México.

Casas, A., & Barbera, G. (2002). Mesoamerican domestication and diffusion. In

P.S. Nobel (Ed.), Cacti: biology and uses (pp. 143-162). Los Angeles, CA:

University of California Press.

Colunga García-Marín, S. P. (1984). Variación morfológica, manejo agrícola y

gradod de domesticación de Opuntia spp. en el Bajío Guanajuatense.

(Master’s Thesis). Colegio de Postgraduados, Chapingo, Mexico.

Crop Profile for Cactus Pear in California. (2000). Washington, DC: United

States Department of Agriculture.

Cummins, J. (1997). Rooting hormones may increase grafting success. Pomona,

2(34). Retrieved from http://www.rooting-hormones.com/cummins.htm

Darwin, C. 1868. The variation of animals and plants under domestication. John

Murray, London, UK.

Diffenbaugh, N. S., Swain, D. L., & Touma, D. (2015). Anthropogenic warming

has increased drought risk in California. Proceedings of the National

Academy of Sciences, 112(13), 3931-3936.

Estrada-Luna, A. A., Lopez-Peralta, C., & Cardenas-Soriano, E. (2002). In vitro

micrografting and the histology of graft union formation of selected species

of prickly pear cactus (Opuntia spp.). Scientia Horticulturae, 92(3), 317-327.

Estrada-Luna, A. A. (1988). Producción de brotes e injertación in vitro de seis

especies de nopal (Opuntia spp) originarias del altiplano Potosino-

Zacatecano (MS Thesis). Colegio de Postgraduados, Campus Montecillo,

Institución en Enseñanza e Investigación en Ciencias Agrícolas.

El Finti, A., El Boullani, R., El Ayadi, F., Ait Aabd, N., & El Mousadik, A.

(2012). Micropropagation in vitro of Opuntia ficus-indica in south of

Morocco. IJCBS, 1, 6-10.

46 46

Evans, G. E., & Rasmussen, H. P. (1972). Anatomical changes in developing graft

unions of Juniperus. Journal of the American Society of Horticultural

Science, 97, 228-232.

Flores, C. A., & Gallegos, C. (1994). The production of prickly pear in the north-

central region of Mexico. In P. Felker and J.R. Moss (Eds.), Proceedings of

the Fifth Annual Texas Prickly Pear Council (13-28). Kingsville, TX: Texas

A&M University.

Frati, A. C., Jiménez, E., & Ariza, C. R. (1990). Hypoglycemic effect of Opuntia

ficus indica in non insulin‐dependent diabetes mellitus patients. Phytotherapy

Research, 4(5), 195-197.

Fuentes, I., Stegemann, S., Golczyk, H., Karcher, D., & Bock, R. (2014).

Horizontal genome transfer as an asexual path to the formation of new

species. Nature, 511(7508), 232-235.

Gahan, P. (2013). Circulating nucleic acids: Possible inherited effects. Biology

Journal of the Linnean Society, 110, 931-948.

Garcıa-Saucedo, P. A., Valdez-Morales, M., Valverde, E.M. Cruz-Hernandez, A.,

& Paredes-Lopez, O. (2005). Regeneration of three Opuntia genotypes used

as human food. Plant Cell, Tissue and Organ Culture, 80, 215-219.

Giusti, P., Vitti, D., Fiocchetti, F., Colla, G., Saccardo, F., & Tucci, M. (2002). In

vitro propagation of three endangered cactus species. Scientia Horticulturae,

95(4), 319-332.

Glenn, E. P., Brown, J. J., & Blumwald, E. (1999). Salt tolerance and crop

potential of halophytes. Critical reviews in plant sciences, 18(2), 227-255.

Griffin, D., & Anchukaitis, K. J. (2014). How unusual is the 2012–2014 California

drought? Geophysical Research Letters, 41(24), 9017-9023.

Gur, A., Samish, R. M., & Lifshitz, E. (1968). Role of cyanogenic glycoside of

quince in incompatibility between pear cultivars and quince rootstocks.

Horticultural Research, 8(2), 113-134.

Hartmann, H. T., Kester, D. E., Davies, F. T., & Geneve, R. (2010). Plant

propagation: principles and practices (8th ed.). Upper Saddle River, NJ:

Prentice-Hall.

Huffmann, M. (2003). Cactus grafting methods. Journal of the Professional

Association for Cactus Development, 5(1), 106-114.

47 47

Inglese, P., Basile, F., & Schirra, M. (2002). Cactus pear fruit production. In P.S.

Nobel (Ed.), Cacti: biology and uses (pp. 143-162). Los Angeles: University

of California Press.

Juárez, M. C. & C. B. Passera (2002). In vitro propagation of Opuntia ellisiana

Griff. and acclimatization to field conditions. BIOCELL, 26(3), 319-324.

Khalafalla, M. M., Abdellatef, E., Mohameed-Ahmed, M. M., & Osman, M.

(2007). Micropropagation of cactus (Opuntia ficus-indica) as strategic tool

to combat desertification in arid and semi arid regions. International

Journal of Sustainable Crop Production, 2(4), 1-8.

Kitchen Culture Technologies Inc. (2010). Plant Tissue Culture for the Serious

Hobbyist, Teacher, Nurseryman and All Plant Lovers. Brush Prairie, WA:

Stiff, Carol.

Köse, C., & Güleryüz, M. (2006). Effects of auxins and cytokinins on graft union

of grapevine (Vitis vinifera). New Zealand journal of crop and horticultural

science, 34(2), 145-150.

Lee, Y. C., Pyo, Y. H., Ahn, C. K., & Kim, S. H. (2005). Food functionality of

Opuntia ficus-indica var. cultivated in Jeju Island. Preventive Nutrition and

Food Science, 10(1), 103-110.

Leifert, C., Ritchie, J. Y., & Waites, W. M. (1991). Contaminants of plant-tissue

and cell cultures. World Journal of Microbiology and Biotechnology, 7(4),

452-469.

Liu, Y., Wang, G., & Li, X. (2011). Michurin’s legacy to biological science.

Journal of biosciences, 36(1), 13-16.

Lohar, D. P., & VandenBosch, K. A. (2005). Grafting between model legumes

demonstrates roles for roots and shoots in determining nodule type and

host/rhizobia specificity. Journal of Experimental Botany, 56(416), 1643-

1650.

Llamoca-Zarate, R. M., Aguiar, L. F., Landsmann, J., & Campos, F. A. P. (1999).

Whole plant regeneration from the shoot apical meristem of Opuntia ficus-

indica Mill. (Cactaceae). Angewandte Botanik, 73(3-4), 83-85.

Maldonado, L. J., & Zapien-Barragan, M. (1977). Nopal en Mexico. Ciencia

forestal, 5(2), 36-53.

48 48

Maria de Fátima, P. S., & Prioli, J. (1996). Micropropagation of Cereus

peruvianus Mill (Cactaceae) by areole activation. In Vitro Cellular &

Developmental Biology-Plant, 32(3), 199-203.

Moghadam, A. R. L., Ardebili, Z. O., & Rezaie, L. (2014). Effect of indole butyric

acid on micrografting of cactus. African Journal of Biotechnology, 11(24),

6484-6493.

Mohamed-Yasseen, Y., Barringer, S. A., Splittstoesser, W. E., & Schnell, R. J.

(1995). Rapid propagation of tuna (Opuntia ficus-indica) and plant

establishment in soil. Plant Cell, Tissue and Organ Culture, 42(1), 117-119.

Mondragón-Jacobo, C., & Pérez-González, S. (Eds.). (2001). Cactus (Opuntia

spp.) as forage. Rome, Italy: Food & Agriculture Organization of the United

Nations.

Moore, R., & Walker, D. B. (1981a). Studies of vegetative compatibility-

incompatibility in higher plants. I. A structural study of a compatible

autograft in Sedum telephoides (Crassulaceae). American Journal of Botany.

68(6), 820-830.

Moore, R., & Walker, D. B. (1981b). Studies of vegetative compatibility-

incompatibility in higher plants. II. A structural study of an incompatible

heterograft between Sedum telephoides (Crassulaceae) and Solanum penellii

(Solanaceae).American Journal of Botany. 68(6), 831-842.

Mudge, K., Janick, J., Scofield, S., & Goldschmidt, E. E. (2009). A history of

grafting. Horticultural Reviews. 35, 437-493.

Murashige, T., & Skoog, F. (1962). A revised medium for rapid growth and bio

assays with tobacco tissue cultures. Physiologia plantarum, 15(3), 473-497.

Sawaya, W. N., Khatchadourian, H. A., Safi, W. M., & Al‐Muhammad, H. M.

(1983). Chemical characterization of prickly pear pulp, Opuntia ficus‐indica,

and the manufacturing of prickly pear jam. International Journal of Food

Science & Technology, 18(2), 183-193.

Shimomura, T., & Fujihara, K. (1978). Prevention of auxin-induced vascular

differentiation in wound callus by surface-to-surface adhesion between

calluses of stock and scion in cactus grafts. Plant and Cell Physiology, 19(5),

877-886.

Sitton, B. G. (1931). Vegetative propagation of the black walnut. Michigan

Agricultural Experiment Station Technical Bulletin. 119.

49 49

Sorce, C., Massai, R., Picciarelli, P., & Lorenzi, R. (2002). Hormonal relationships

in xylem sap of grafted and ungrafted Prunus rootstock. Scientia

Horticulturae, 93, 333-342.

Tous, J., & Ferguson, L. (1996). Mediterranean fruits. In J. Janick (Ed.), Progress

in New Crops (pp. 416-430). Arlington, VA: American Society for

Horticultural Science Press.

Turner, R. M., Alcorn, S. M., & Olin, G. (1969). Mortality of transplanted saguaro

seedlings. Ecology, 50(5), 835-844.

Van Hooijdonk, B.M., Woolley, D.J., Warrington, I.J., & Tustin, D.S. (2010).

Initial alteration of scion architecture by dwarfing apple rootstocks may

involve shoot–root–shoot signaling by auxin, gibberellin, and cytokinin.

Journal of Horticultural Science and Biotechnology. 85(1), 59-65.

Williams, J. H. (1996). A great and shining road: The epic story of the

transcontinental railroad. Lincoln, NE: University of Nebraska Press.

Zhou, X., & Liu, Y. (2015). Hybridization by grafting: A new perspective?

HortScience, 50(4), 520-521.

Fresno State

Non-Exclusive Distribution License

(to archive your thesis/dissertation electronically via the library’s eCollections database)

By submitting this license, you (the author or copyright holder) grant to Fresno State Digital

Scholar the non-exclusive right to reproduce, translate (as defined in the next paragraph), and/or

distribute your submission (including the abstract) worldwide in print and electronic format and

in any medium, including but not limited to audio or video.

You agree that Fresno State may, without changing the content, translate the submission to any

medium or format for the purpose of preservation.

You also agree that the submission is your original work, and that you have the right to grant the

rights contained in this license. You also represent that your submission does not, to the best of

your knowledge, infringe upon anyone’s copyright.

If the submission reproduces material for which you do not hold copyright and that would not be

considered fair use outside the copyright law, you represent that you have obtained the

unrestricted permission of the copyright owner to grant Fresno State the rights required by this

license, and that such third-party material is clearly identified and acknowledged within the text

or content of the submission.

If the submission is based upon work that has been sponsored or supported by an agency or

organization other than Fresno State, you represent that you have fulfilled any right of review or

other obligations required by such contract or agreement.

Fresno State will clearly identify your name as the author or owner of the submission and will not

make any alteration, other than as allowed by this license, to your submission. By typing your

name and date in the fields below, you indicate your agreement to the terms of this

distribution license.

Embargo options (fill box with an X).

Make my thesis or dissertation available to eCollections immediately upon

submission.

Embargo my thesis or dissertation for a period of 2 years from date of graduation. After

two years, I understand that my work will automatically become part of the university’s

public institutional repository.

Type full name as it appears on submission

Date

Jacob Hurst

December 8, 2016

x