Nghiên cứu kỹ thuật nhân giống vô tính cây đu đủ carica papaya l cv ‘tainung no 2’ and ‘red lady’

Nghiên cứu kỹ thuật nhân giống vô tính cây đu đủ carica papaya l cv ‘tainung no 2’ and ‘red lady’ Nghiên cứu kỹ thuật nhân giống vô tính cây đu đủ carica papaya l cv ‘tainung no 2’ and ‘red lady’ Nghiên cứu kỹ thuật nhân giống vô tính cây đu đủ carica papaya l cv ‘tainung no 2’ and ‘red lady’ Nghiên cứu kỹ thuật nhân giống vô tính cây đu đủ carica papaya l cv ‘tainung no 2’ and ‘red lady’ Nghiên cứu kỹ thuật nhân giống vô tính cây đu đủ carica papaya l cv ‘tainung no 2’ and ‘red lady’ Nghiên cứu kỹ thuật nhân giống vô tính cây đu đủ carica papaya l cv ‘tainung no 2’ and ‘red lady’

國立屏東科技大學熱帶農業暨國際合作系 Department of Tropical Agriculture and International Cooperation National Pingtung University of Science and Technology

博士學位論文 Ph.D Dissertation

台農 2 號與紅妃番木瓜(Carica papaya L.)無性繁殖之研究

Studies on asexual propagation techniques of papaya (Carica

papaya L.) cv ‘Tainung No.2’ and ‘Red Lady’

指導教授 Advisors: 顏昌瑞 博士 (Chung-Ruey Yen, Ph.D.)

謝清祥 博士 (Ching-Hsiang Hsieh, Ph.D.)

研究生Student: 阮文鴻 (Nguyen Van Hong)

中華民國 107 年 10 月 30 日

October 30, 2018

no starch (Sampson 1986 ) Papain, whose proteolytic action is similar to that

of pepsin and trypsin, is employed as a meat tenderizer in applications in the food industry, as well as in the textile, pharmaceutical, and cosmetic industries (Villegas 1997, Su 2009)

Papaya is one of the most economically important fruit crops in many tropical and subtropical countries In 2016, total areas for fruit cultivation were 441,964 ha in the world which produced 13,050,749 tonnes (t) of fruit (Faostat 2017) In Taiwan, papaya is one of the top ten fruits of production (Fig.5) In 2015, papaya area harvested and production were 2,500 ha and 115,115 tonnes (Faostat 2017) Recently, the papaya production is affected by destructive diseases, specially, papaya ringspot (PRS) PRS is one the most destructive diseases of papaya and occurs in every region where papaya is grown It has been reported to be a major limiting factor for commercial papaya production particularly in Hawaii, areas of Thailand, Taiwan, India, Mexico, Bangladesh, the Philippines, and the southern region of China (Chang 2003, Jayavalli 2011)

Papaya is a polygamous species with many forms of inflorescences The species has three sex types: Staminate, pistillate and hermaphrodite (Dinesh 2001, Paull 2011) Papaya is one of the few fruit crops still mostly

propagated by seed for commercial production Papaya seedlings propagated from seed is hindered by problems because of the sex reversal, inherent heterozygosity and dioecious nature of the crop (Teixeira 2007, Clarindo 2008) In the commercial plantations of most producing countries, male plants are useless and only hermaphrodite individuals are agreed by growers (Usman

2002, Hsu 2012) However, it was found that undesirable male plants prevail

as high as 30% and sometimes over 50 % of trees planted in papaya fields (Jordan 1983) So, in actual commercial production, three or four seedlings are planted at each position, and when their sexes are determined, only hermaphrodites are kept There are cases in which none of them are hermaphrodites In addition, the plants grown from seeds show considerable variations in disease susceptibility, fruit quality, and yield (Reuveni 1990, Allan 1995, Teixeira 2007)

The main advantage of vegetative propagation is the certainty of keeping the characteristics of the mother plant (Hartmann 2002, Hartmann 2011) It had been reported the possibility of developing materials highly productive and resistant to diseases, which can be spread safely keeping intact the characteristics of the papaya mother plants through asexual propagation (San Jose 1988) Additionally, one can reduce transmissible diseases by choosing mother plants carefully The case of gynodioecious cultivars, the bisexual types which produce fruits with shape, size, and flavor are preferred

to round fruits of female plants as they fetch premium price in the market (Reuveni 1990, Teixeira 2009) Up to now, asexual propagation techniques, such as rooting of cuttings, grafting and micropropagation have been successful in papaya cultivars (Airi 1986 , Ramkhelawan 1998 , Teixeira

2007, Chong 2008 , Wu 2012, Setargie 2015)

The success of assexual propagation by cutting, grafting, tissue culture depends on numerous factors, among them the zone environment, the material and technique application, and the genotype (Hartmann 2002, Soundy 2008, Hartmann 2011, Mabizela 2017) In addition, the results can not apply to all

varieties and in all climatic conditions So, scientists need conduct much more research on each variety under certain conditions

In Taiwan, the papaya is mainly propagated by seed and by a method

designed to reduce damage from viruses of insects To date, there is hardly

any information on cuttings, grafting and tissue culture propagation of the hybrid papaya cultivars ‘Tainung No.2’ and ‘Red Lady’ The ‘Tainung No.2’ papaya is the major cultivar with 90% of growing area and ‘Red Lady’ papaya is potentially one for spreading with fruit weigh of 1.5-2 kg, good fruit quality (flesh is thick, red, with 13% sugar content, and aromatic) and preferred by the local market (Agriculture and Food Agency, Council of Agriculture, Executive Yuan, R.O.C) So, on purpose of cloning good quality papaya varieties, we conducted researches on propagation of two papaya varieties (‘Tainung No.2’ and ‘Red Lady’) by grafting, cutting and tissue culture The aims of this study were to investigate the effects of grafting, cutting and micropropagation techniques on commercial asexual propagation

in 'Tainung No.2' and 'Red Lady' papaya The specific objectives were:

• Research on grafting propagation of ‘Tainung No.2’ and ‘Red Lady’ papaya

• Research on cutting propagation of ‘Tainung No.2’ and ‘Red Lady’ papaya

• Research on tissue culture propagation of ‘Tainung No.2’ and ‘Red Lady’ papaya

LITERATURE REVIEW

1 General of papaya plant

Papaya (Carica papaya L.) is a popular fruit native to tropical America

Papaya plant is grown for its melon-like fruit It is a herbaceous perennial plant, bearing fruit continuously at the leaf axils spirally arranged along the single erect trunk The papayas have common names, such as papaya, papaw

or pawpaw, papayer (friench), melonenbaum (German), lechosa (Spanish), mamao (Portuguese), mugua (Chinese), and dudu (Vietnamese) (Paull 2011)

1.1 Taxonomy

Carica Papaya L., is the most important economic fruit, belongs to the Carica Genus, Caricaceae family Caricaceae is a small family of

dicotyledonous plant with five genera of tropical American origin (Carica,

Jarilla, Jacaratia, Horovitzia and Vasconcella) and one from equatorial Africa (Cylicomorpha) (Paull 2011) There are 32 species described with distribution: Carica, 1 species, Jarilla, 3 species, Jacaratia, 5 species,

Horovitzia, 1 species, Vasconcella, 20 species, and Cylicomorpha, 2 species

Carica and Vasconcella species are dioecious, except for the

monoecious Vasconcella monoica (Desf.) and some Vasconcella pubescens and the polygamous C papaya Most species are herbaceous, single-stemmed

and erect (Paull 2011)

1.2 Origin, distribution and production

Upto now, scientist has not found Carica papaya wild in nature It is evidenced in distantly relation to the Vasconcella species by isozyme and

AFLP analysis (Paull 2011) It is believed that Carica papaya is native to tropical America, Its origin region is southern Mexico and neighbouring Central America (Morton 1987) In the 16th century, spanish took papaya to the Caribbean and South East Asia In the accounts of 18th century, seeds of

papaya had been taken from the Caribbean to Malacca and on to India (Paull 2011) Subsequent historical records indicate that from Malacca or Philippines the papaya distribution continued throughout Asia and to the South Pacific region The factors such as a large number of the seeds in the fruit and their long viability have contributed to the wide geographical distribution of the fruit (Paull 2011)

In the past decade, papaya has attained great popularity because it can

be intensively cultivated, its rapid returns and the increased demand for the fresh fruit as well as its processed products Papaya is commercially cultivated between 23o North and 32o South latitude (Paull 2011), an area which includes many tropical and sub-tropical countries of the world From

2006 to 2016, Papaya area harvested and production had been developing quickly in the world (Figure (Fig.) 1) The highest papaya production was obtained in Asia (49%) followed by Americas (37.7%) and by Africa (13.2%) The lowest production (0.2%) is recorded Oceania (Fig.2) Top ten countries of papaya production (2006-2016) are India, Brazil, Indonesia, Negeria, Mexico, Dominican Republic, Democratic Republic of the Congo, Kenya, Thailand, Colombia, Philippines (Fig.3) (Faostat 2017)

Figure 1 Production of papaya in the world 2006-2016 (Source: Faostat,

2017)

0 50000 100000 150000 200000 250000 300000 350000 400000 450000 500000

0 2000000 4000000 6000000 8000000 10000000 12000000 14000000

Figure 2 Production share of papaya by region (Source: Faostat, 2017)

Figure 3 Top papaya production countries (Source: Faostat, 2017)

Papaya production in Taiwan

In 17th century, papaya travelled from the west Indies to Asia where at the end of the Qing Dynasty it was introduced to Taiwan and be developed forcefully upto now In 2016, papaya area harvested and production achieved 2,584 ha and 118,661tonnes, respectively (Faostat 2017) Periods of 1994-

2016, papaya area harvested and production were varied in Taiwan and they were increasing trend in 2014-2016 (Fig 4) Papaya is in top 10 fruit growing

Asia 49%

Americas 38.7%

Africa 13.2 %

Oceania 0.2 % Asia Americas Africa Oceania

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

Figure 4 Area harvested and production of papaya in Taiwan (Source:

Faostat, 2017)

Figure 5 Fruit production in Taiwan in 2013 (Source: COA, TIER, 2014)

1.3 Characteristic of papaya plant

* Stem

The papaya plant is a large, mono-axial herbaceous plant with an erect stem terminating with a crown of large leaves and can attain by 9 m heights (Fig 6) Although there are occasional lines or cultivars that produce and

Wax Apple 4%

Pear 4% Papaya

5%

Betel nut 5%

Guava 7%

Mango 8%

Banana 7%

Pineapple 16%

Citrus 20%

Other fruits 24%

0 500 1000 1500 2000 2500 3000 3500

0 20000 40000 60000 80000 100000 120000 140000 160000

abundance of lateral branches, especially during the juvenile period, the main stem normally growth of the axillary branches does occur when the trees become 3-5 years old (Paull 2011) The stem is semi-woody and hollow and a major site of starch storage The bark is smooth, grayish, with large and prominent leaf scars When the stem is wounded, a thin milky sap oozes from the wound (Fig 6)

After transplanting, shoot growth is initially slow, though considerable root growth is taking place, extending out well beyond the canopy drip line Stem growth is then rapid up to flowering, increasing in circumference up to 2

mm per day (Paull 2011) Growth rate peaks at flowering then declines as the tree starts bearing The rate of growth is influenced by nitrogen and phosphorus supply, irrigation and temperature (Paull 2011)

Figure 6 Vegetative parts of the papaya plant (a) Cross section of a

1-year-old papaya stem: periderm (pe), fiber sheath (fs), phloem (ph), cambium (c), xylem rays (xr), pith (p) (b) Leaf lamina and petiole (c) Longitudinal section of a 3-month-old papaya stem showing hollow pith cavity (d) Longitudinal section of a 1-year-old papaya stem showing complete pith cavity (e) Stem of a 1-year-old papaya plant showing conspicuous petiole scars

* Leaves

A cluster of leaves occurs at the apex of the plant and along the upper part of the stem and makes up the foliage of the tree (Fig 6) New leaves are constantly formed at the apex and old leaves senesce and fall Leaves are palmately lobed with prominent veination and can measure 40-50 cm or more

in diameter and have an individual leaf area of 1625 cm2, with 15 mature leaves per plant (Paull 2011) In the tropics, new leaves appear two to three a week (Chan 1984) Petioles are cylindrical, hollow and length of 60 – 90 cm, depending upon the cultivar The most recently matured leaf’s fresh weight (about 10th leaf from 2.4 cm juvenile leaf) varies from ca 50 to 170 g The leaf petiole dry mass increases at a rapid rate until flowering the increases more

slowly, peaking after fruit bearing starts (Paull 2011)

* Floral organization and flowers

Papaya has three types of distinctly different flowers, male, female and hermaphrodite, which give rise to fruits (Bose 1990) (Fig 7) The flowers are found in the auxiliary pendulous in the inflorescence Male flowers are yellowish, 2-4 cm long with petals fused to form a long tube, with 10 fertile stamens and a rudimentary ovary (Fig.7 A, D) Female inflorescence is much shorter, 3-4cm long and sits alone or in small groups in leaf axils Female flowers are larger, usually white or cream in color, with five free petals and a large ovary with 5 fan shaped stigmas without stamens (Fig 7 C, F) Hermaphrodite flowers have either 5 or 10 stamens and a prominent ovary (Fig 7 B, E)

Five major floral structures namely pistillate, pentandria, elongate,

staminate and intermediate have been identified of papaya (Bose 1990)

Figure 7 Papaya flowers with one petal removed to show internal parts (A–C)

and inflorescences (D–F) (A) Staminate flower showing stamens (st), pistillode (pi) and corolla tube (ct) (B) Perfect flower showing

st, ct, stigmata (sa), petal (p) and an elongated ovary (o) (C) Pistillate flowers showing sepals (sp), petals and round ovary (o) (D) Long male inflorescence with dozens of staminate flowers (E) Andromonoecious cyme showing one dominant perfect (pf) and five secondary staminate flowers (sf) (F) Female cyme with three pistillate flowers

* Fruits

Papaya fruits are usually born auxiliary on the main stem often as single and rarely in small clusters They are generally melon-like (oval to nearly round) or somewhat pyriform or elongated club shaped with a waxy skin The flesh is yellow – orange to salmon at maturity and pale yellow in unripened ones The shapes of the fruits vary according to the sex and the variety of papaya qualifying sex as an important trait in papaya (Parasnis 1999)

The fruit is rich in carbohydrates, fats, proteins, fiber minerals and vitamins and is also a good source of iron calcium, vitamins A, B and C The unripe hard green fruit contain white latex, which is rich in papain (Rubens Monti 2000) The yellow, orange or reddish succulent flesh in the ripe fruit underneath the thick wall of the fruit is aromatic, juicy and sweetish and harbors seeds lightly by soft, white, fibrous tissue

* Seeds

The papaya seeds are black, corrugated, ovoid, peppery and coated with

a transparent gelatinous coat The structure of the seeds concludes the seed coat, endosperm, embryo

Figure 8 Seed and seedling A Mature dried seeds, T.S (transverse section)

and L.S (longitudinal section) B Endosperm cells C Ovule wall

of developing seed (em) embryo, (en) endosperm, (mt) mesotesta ridge, (vb) vascular bundle, (ii) inner integument, (oi) outer integument, (sad) sarcotesta (Source: Fisher 1980)

Seed coat: The outer region is fleshy and becomes a gelatinous

sarcotesta at maturity (Fig 8C) It is derived from the multiple outer epidermis of the outer integument of the ovule The mesotesta is compact, consisting of a series of sculptured, spongy, and hydroscopic longitudinal ridges, derived chiefly from subepidermal layers of the outer integument (Fig 8A) The inner epidermis of the outer integument remains unchanged except for development of druses The inner integument produces thin, inner, sclerotic layers of the seed coat with the inner epidermis tanniniferous and

subepidermis fibrous The funicle is stout, its head occasionally enlarged and fleshly as a short aril (Fisher 1980) The funicular vascular bundle extends into the inner integument at the chalazal end where it subdivides

Endosperm: Cells are thin-walled with abundant oil and aleurone

grains; starch is absent at maturity (Fig 8B )

Embryo: The embryo is straight and median with ovoid and flattened

cotyledons (Fig 8A )

* Root system

Young roots show well-differentiated epidermis, cortex, and endodermis, enclosing an exarch vasculature in which six xylem and six phloem poles alternate Cambium formation in a concentric ring triggers secondary growth and root thickening while maintaining succulence The papaya root is predominately a non-axial, fibrous system, composed of one or two 0.5–1.0 m long tap roots Secondary roots emerge from the upper sections and branch profusely (Fig 9 a, b) Healthy roots are whitish cream and no laticifers have been observed in them (Marler 1997, Carneiro 2009)

Figure 9 Root system (a) Side view of an excavated 5-month-old papaya root

system, showing the main and secondary roots (b) Upper view of the same root system, showing horizontal distribution of secondary roots

Phenotypic plasticity of roots is high Size, number, distribution, and orientation of plant roots adjust readily across the soil profile, to various soil conditions, and throughout the life of the plant (Marler 1997, Carneiro 2009)

Plants dependent on mycorrhizas for their nutrition and benefit from soil mulching and appropriate drainage that facilitate biotic interactions in the rhizosphere and water and nutrient uptake Mycorrhizal interactions of male and female papaya plants may differ: females seem more responsive to changes in soil fertility and readily adjust mycorrhizal colonization accordingly (Vega-Frutis 2009)

1.4 Sex characteristics of papaya

Papaya crop is polygamous in nature The sex of dioecious papaya plants only can be defined after they attain reproductive maturity (6–8 months) (Bose 1990) Normally, 50% of the population in a field is composed

of unfruitful male plants and almost 45% of these have to be uprooted at the flowering stage (Parasnis 1999) This unnecessary cultivation of unwanted males leads to wastage of resources, which can be avoided if the sex of the plant is determined at juvenile stage Morphological and cytological studies conducted so far have failed to differentiate between the various sex forms of papaya (Parasnis 1999) Papaya exhibits wide morphological and biological diversity of sex types with prominent specific characters The papaya plants can be either dioecious with male or female flowers occurring in separate plants or monoecious with male and female parts falling in the same flower (Yon 1994)

Three sex forms (female, male, and hermaphrodite) are regulated by an incipient X–Y chromosome system Papayas can be either dioecious (with male and female plants) or gynodioecious (with hermaphrodite and female plants) Several studies suggested that the Y chromosome contains a small specific region that controls expression of male (Y) or hermaphrodite (Y h ) types Female plants are of the XX form All combinations among the Y

and/or Y h chromosomes are lethal; therefore, the male and hermaphrodite types are heterozygous (XY and XY h , respectively) (Ming 2007)

In papaya the change of sex occurs in some trees at high temperature, where short stalked male flowers are produced instead of usual perfect flowers (Chan 2009) Male or bisexual plants changing completely too female plants after being beheaded and some “all male” plants occasionally producing small flowers with perfectly pistils leading to abnormally slender fruits are also instances of change of sex in papaya (Sujitha 2012)

Chay-Prove (Chay-Prove 2000) and OECD (Organization for Economic Cooperation and Development 2003) showed that: Changes in functional gender in response to environmental viriables have been used advantageously in papaya breeding programs and to help select the most appropriate varieties for commercial cultivation in particular regions

* The environmental factors affect papaya sex expression

The basic sex types in papaya are genetically determined The pistillate

or female is phenotypically very stable However, certain male and hermaphrodite trees have been known to undergo sex reversal under the influence of various environmental changes (Ming 2007 )

Temperature

Cool temperatures in the winter months appear to promote more femaleness in hermaphrodite trees (Allan 1987) Cooler temperature reduces the number of stamens, as described previously is brought about by fusion of the stamens to the ovary (Jain 2009) The warm temperatures tend to promote the production of hermaphrodite flowers resulting in the sterility of the trees When such conditions persist over a length of time, a production gap along the trunk is clearly visible

Allan et al (Allan 1987) in a study on environmental effects on clonal female and male papaya plant found that cool night temperatures of about 12

oC and short daylengths appeared to be critical in causing sex reversal from the sterile staminate to fertile, elongata type hermaphrodite flowers

Moisture

Moisture levels probably affect the growth and development of the trees and indirectly bring about the reversal in sex (Jain 2009) High moisture regime promotes femaleness in papaya For hermaphrodite plant, consistently high moisture levels will promote the production of hermaphrodite flowers with reduction of stamen number and low soil moisture regime tend to produce more elongate type hermaphrodite flowers (Jain 2009)

Nitrogen

Application of nitrogen promoted greater tendency towards femaleness

in hermaphrodite trees ( Jain 2009) Manipulation of nitrogen levels applied to the plants able to control the expression of sex (Teixeira 2009)

Growth regulators

Ethylene promotes femaleness in papaya (Pet Roey 2014) Papaya seedlings of a dioecious cultivar treated by Ethephon (a conversion from ethylene) exhibited a significantly higher percentage of female trees at maturation This percentage was over 90% when applications were continuously given at 15-day or 30 day intervals until emergence of flowers

2 Propagation in papaya

2.1 Sexual propagation in papaya

Papaya propagation by seed plays a particularly important role in the production of plantlets and have been considering as a major method in commercial propagation (Senthilkumar 2014, Abdel 2016, Omar Schmildt

2016) Seeds can be obtained from natural pollination or artificial pollination Seeds are produced in seedling centers by controlling process in parental pollination Seeds of some papaya verities are easily reproducible while some require elaborate pollination procedures

The ratio of female, hermaphrodite and male offspring are predictable,

as summarized in Table No 1 (Jain 2009) and as follows:

(1) Pistillate flowers pollinated by staminate flowers give equal numbers of male and female progeny

(2) Pistillate flowers pollinated by pollen from bisexual flowers give equal numbers of female and bisexual progeny

(3) Bisexual flowers either self or cross-pollinated with other bisexuals give a ratio of one female to 2 bisexual

(4) Bisexual flowers pollinated by staminate ones produce equal numbers of female, male and bisexual progeny

Table 1 Summary of gender ratios following pollinations between male (M),

Felmale (F) and bisexual (B) C papaya gender forms

Females

No of resulting Males

No of resulting Bisexuals

soak them for 24 hours The sink seed can be used for germination and the remaining seeds is continued to soak in fresh water for more 24 hours Then, the sink seeds are used for germination and discard the rest (bad seeds) During the soaking phase, we must ensure to use very clean water

Once the seeds have been saturated with clean water they can be germinated in several ways The two most common methods to germinate seeds are by using sterile potting medium or a cloth sling Either way, the medium containing the seed needs to be sterile and kept moist at all times The optimum temperature for germination is about 25 oC Seeds germinate in

2 to 5 weeks

Some methods such as removing seed sarco-testa (Chow 1991), soaking in growth regulators (Tawfik 2002) or in potassium nitrate (Montejo 2002) or in magnetized water (Espinosa 1997) or in leaf extract and powder (Ananthakalaiselvi 1998), putting seeds in temperature extremes (Salomao

2000, Wood 2000), treating matri-conditioning (Andreoli 1993), and in vitro germination (Bhattacharya 2001) were successful to improve papaya seed germination

Papaya plants propagate from seeds only can be defined after they attain reproductive maturity (6–8 months) (Bose 1990) So, to ensure plant

density in the production plantation, growers plant three or four seedlings at each position before extracting tree having unwanted sex characteristics

* Advantages and disadvantages of propagation by seed

Advantages of propagation by seed

Seed propagation is a common and conventional method of propagation

in papaya It is very simple and easy method of propagation that does not much advanced or complicated techniques Farmers generally collect fruits of good quality from their orchards and the extract seeds for subsequent plantings So farmers or growers can apply it in their gardens or orchards According to recommendation of seed manufacture in market, the germination rate is over 75% (Chauhan 2014) In cases of other propagations that are difficult to implement or ineffective due to lack of facilities and equipment for in-vitro culture, and inadequate materials for cutting propagation, seed propagation seems to be a suitable and effective tool for seedling multiplication

The cost of seedlings from seeds are cheaper than cost of seedlings from cuttings, grafting and tissue culture According to my investigation in Taiwan, the cost of a seedling propagated by seeds is about 3-15 TWD (Taiwan dollar) belong to variety and planting season and be cheaper than cost of seedling propagated by asexual method (25-50 TWD with grafting)

Seedlings propagated by seeds have strong roots It is advance for growth of plant when papaya plant in the field The seedling plants are long lived and are resistant to water stress Specially, strong root system of papaya helps plant coping with frequently storm in sub-tropic and tropic

Hybrid seed production needs to be developed by this method, especially creating new hybrid generation

Transmission of viruses can be prevented by seedling method In polyembryonic seeds, the apospory form, which maintains the genetic systems similar to their mother plants, is a valuable material source for seedling or micro-propagation due to free diseases

Due to more efficiency of root activities, seed propagation is used to produce root stocks for budding and grafting Seed can be transported and stored more convenient and for longer time for propagation

Disadvantages of propagation by seed

Propagation is hindered with problems associated with inherent heterozygosity and dioecious nature of the plant (Manshardt 1992, Bhattacharya 2001, Paull 2011) Seeds derived from open pollinated flowers can produce plants with considerable variation in sex types (a mix of male, female and hermaphroditic plants) which is highly undesirable when this results in variation in disease susceptibility, fruit quality and yield (Allan

1995, Teixeira 2007) Heterozygous and a cross pollinated crop, sexual propagation has resulted in immense variation among populations for growth duration, yield, size, shape, quality of fruit and disease susceptibility leading

to production of non-true-to-type plants (Panjaitan 2007) The disadvantage of dioecious varieties is that unproductive male plants prevail as high as 30% and some times over 50% of the total in commercial plantation (Jordan 1983) This makes the wastage of money, time and labours for the problem of true-sex plant density leads to a reduction in production efficiency

Marin and Silva (Marin 1996) stated that the possibility of maintaining original characteristics of the parent plants of papaya does not occur in the conventional system of production where seeds are harvested in majority of cases, from commercial open pollinated orchards

In addition, as the same as other fruit crops, seedlings propagated by seeds have a more juvenile period than those propagated by vegetative propagation (Hartmann 2011)

The use of seeds for papaya production has both positive and negative facets Numerous seeds are available from one papaya fruit, but seed

germination can be slow and sporadic (Perez 1980) Reyes et al (Reyes 1980)

and Yahiro and Yoshitaka (Yahiro 1982) isolated “germination inhibitors” in the sarcotesta and inner seed coat but not in the embryo and endosperm In addition, the occurrence of inhibitory substances present in the aril, extraction and conservation of seeds may cause germination loss in relatively short periods (Couto 1983)

2.2 Asexual propagation in papaya

Vegetative propagation of plants is their reproduction from vegetative organs: stems, roots, leaves, buds, even single cells Through vegetative propagation or cloning, show exact copies of the ‘mother plant’ are produced,

a process that can – in theory – be repeated indefinitely The phenomenon of vegetative propagation is based on the ‘omnipotence’ of plant cells, meaning that any plant cell, unlike most animal cells, has the potential to regenerate into a functioning organism Vegetative propagation as opposed to sexual propagation (by seed) offers a range of benefits in tree domestication as well

as in conservation efforts

Papaya is one of the fruit crops, which to date, have defied attempts to clonally propagate by vegetative means In current papaya asexual propagation, main applied methods are cutting, grafting, and tissue culture (Paull 2011)

As other crop asexual propagation, vegetative propagation methods in papaya produce new plants from vegetative parts of the original plant, such as the buds, stems, and leaves etc In asexual propagation, only a single parent is

required and thus t is no need propagation mechanisms such as pollination, cross pollination etc. In this process, no exchange of genetic information takes place as the offspring is formed through material of a single parent (Hartmann 2002, Teixeira 2007, Paull 2011).Thus the resultant plants formed contain the genetic material of only one parent, so they are essentially clones

of the parent plant

Vegetative propagation becomes imperative for the production of standard varieties from the most outstanding local types, those developed through breeding programs

There are many environmental factors affecting papaya asexual propagation such as light (irradiance, daylength, light quality), temperature, gases and gas exchange, media and nutrition (Muhamad Hafiz 2014, Ofori-Gyamfi 1998, Tchinda 2013, Wendling 2002, De Oliveira 2017, John 1997, Loach 1986, Adugna 2015, Jordan 2010, Muhammad 2006, Owuor 2009, Baltunis 2010, McIvor 2014) In addition, papaya genotype is the factor make large differences to vegetative propagation process (Husen 2003, Teixeira

2007, Baltunis 2010, Rambaran 2013, McIvor 2014, Setargie 2015)

* Advantages and disadvantages of papaya asexual propagation:

Advantages of asexual propagation

One advantage of asexual propagation is that the sex is determined from selection mother plant (plant material propagation) Plantlets formed through asexual process are genetically identical, useful traits can be preserved among them (Hartmann 2011, Paull 2011)

The use of vegetative propagation techniques can produce a large number of true to type high quality planting material is an essential requirement in papaya cultivation (Teixeira 2007, Hartmann 2011)

The plants also bypass the immature seedling phase and therefore reach the mature phase sooner in vegetative propagation (Hartmann 2002) Plants grown through vegetative propagation bear fruits early, lower fruiting height, greater fruit weight, longer cropping span and improved yield over plants grown from seeds (Li- Hung 2004, Allan 2013)

It was reported that the possibility of developing materials highly productive and resistant to viruses, which can be spread safely keeping intact the characteristics of the mother plants of papaya through asexual propagation (San Jose 1988, Dhekney 2016)

According to Reuveni et al (Reuveni 1990) the clonal propagation technique of selected mature female and male plants in papaya is highly desirable for commercial practice, especially in subtropical areas, as the dioecious lines exhibit considerable variation in shape, size and flavor of the fruit and disease susceptibility

Injured plants can be recovered or repaired through techniques involved

in asexual propagation (Bose 1990, Ramkhelawan 1998 , Hartmann 2002, Abdel 2016, Dhekney 2016)

Asexual propagation methods have been applied significantly in papaya breeding They help clone propagation only male or female in crossbreeding (Chan 2009) and in embryo rescue (Magdalita 1996)

Disadvantages of asexual propagation

Papaya is one of the fruit crops, which to date, have defied attempts to clonally propagate by vegetative means Like most other higher plants papaya has an indeterminate mode of growth in which the leaf axils contain subsidiary meristems, each of which is capable of growing into a shoot that is identical to the main axis Axillary branching under natural conditions is inhibited by apical dominance This natural growth habit of papaya plant

chemicalinduction of branching has been accomplished to avail planting materials for vegetative propagation (Allan 1995, Tawfik 2002)

Vegetative propagation methods for some papaya cultivars have been described in the literature (Allan 1995, Ramkhelawan 1999, Tawfik 2002) but none is suitable for rapid production of large numbers of clonal transplants of desired papaya cultivars for commercial plantations Furthermore, these techniques are often slow, time consuming, tedious, impractical, need high technical know-how, and are not widely known by growers in many papaya growing countries (Ramkhelawan 1999)

Vegetative propagation of trees is easily achieved when juvenile sources of propagation materials are used A major limitation is the need to use juvenile tissues from young trees since maturation is usually associated with a declining regenerative capacity(Babu 2002):

In addition, as propagation of other crops, diversity is lost in asexual propagation which is the main reason behind occurrence of diseases in future plant species; papayas produced through this asexual propagation (cutting and grafting) have shorter life-span than those grown through sexual process; Papaya tree involved in this process are less likely to resist pests and diseases (Hartmann 2011)

Grafting methods are labor intensive and need good training before they can be successfully applied However, grafting has become the most used way to improve high-value fruit trees Grafting combines two independent individual plants, and produces one functioning individual The process of graft union formation is regulated by a complicated balance of plant hormones and enzymes Five stages have been defined (Hartman 1986): Lining up the vascular cambiums of rootstock and scion; Wounding response, callus bridge formation, wound-repair xylem and phloem, and production of secondary xylem and phloem

The important requirements for successful grafting operation, producing a plant:

• The stock and scion must be compatible

• The cambial region of the scion must be placed in intimate contact with that of the stock

• The grafting operation must be done at a time when stock and scion are in the proper physiological stage

• Immediately after the grafting operation is completed, all cut surfaces must be protected from desiccation

• Proper care must be given the grafts for period of time after grafting

The methods of plant grafting:

There are many methods of grafting, some very specific for a particular species According to Hartmann et al (Hartmann 2002), there are many methods of grafting such as whip grafting, splice grafting, side grafting, cleft grafting, wedge grafting, bark grafting, inarching, and bridge grafting Cleft grafting is a good choice for species that callus readily produced and is one of the popular methods used for papaya propagation Steps in cleft grafting were presented following:

• Choose a straight part of the rootstock shoot, if possible the same diameter as the scion you have

• Make a cut straight down the middle of the rootstock shoot, to a depth of about three to four times the diameter of the scion (Fig 10A)

• Cut the base of the scion to a long wedge (Fig 10.B)

• Insert most of the length of the wedge into the slit in the rootstock Leave exposed the semi-elliptical ‘church window’ areas of cut surface at the top of the wedge These areas serve as a source of callus to help heal between the scion and the cut top of the rootstock

• Align the wedge in the cleft so that the cambial areas of scion and stock match If the scion and stock wood diameters are the same, centre the scion in the cleft If there is a discrepancy in sizes, match the wood/bark junctions of stock and scion on one side by careful inspection as you slide the wedge into the cleft (Fig 10 C)

• Wrap tightly, trying to maintain the position of the scion in the stock When wrapping, you may find it easier to start at the top of the cleft and then work down

• Wrap carefully with PVC tape, covering the top of the rootstock and the exposed cut areas, and for dormant scions you will only need to seal any cut tip Protect a green scion from drying by covering it, as described for the splice graft

Figure 10 Cleft grafting

* Achievement in papaya grafting research

Grafting has been applied successfully by Airi et al (Airi 1986 ) who cleft-grafted scion shoots from cultivars Co-1 and Honey Dew onto uniformly established seedlings T budding also can be used, but the success rate is poorer than with cleft grafting

In Malaysia, some growers use grafting in the orchard to supersede female-fruiting trees of the cultivar Eksotika (Cheah 1993)

All the combinations were compatible and produced vigorous plants Grafting and inarching of promising papaya hybrids and inbreds onto C cauliflora, a wild resistant to PRSV-P was found to delay the symptom expression in papaya (Villegas 1997)

Ramkhelawan et al (Ramkhelawan 1998) conducted studies to develop simple methods of propagation of papaya in vivo, thus providing growers a

wide choice in methods of production of standard varieties Among various propagation methods, terminal wedge grafting was clearly superior to the chip budding and side grafting, with success rates of 100%, 85% and 75% respectively and good growth performance in the field

The effect of grafting on the fruiting of Tainung No 2 and Tainung No

5 papaya varieties revealed that grafted plants had a tendency of being shorter than ungrafted seedlings; they did not show incompatibility between scion and rootstock and yielded better than ungrafted papaya trees (Weng 1999)

Fitch Maureen et al (Fitch Maureen 2005) stated that the clonally

propagated papaya plants were significantly shorter

Phenology and production of Carica papaya ‘Honey Gold’ under cool subtropical conditions were reported by Allan (Allan 2007) Vegetative propagation of selected, red fleshed hermaphrodite types ensured the production of fruits of outstanding quality for discerning markets

Chong et al (Chong 2008) reported the grafting success (about 80%)

through cleft method in ‘Eksotika’ papaya at nursery stage They stressed the advantage of grafted papaya trees as they bear fruits much lower and earlier and are dwarf in stature with longer economic life cycle There is also potential in utilizing rootstocks for tolerance to ‘wetfeet’ and soil-borne diseases A better approach of obtaining 100% hermaphrodite stand by cleft grafting papaya seedling using healthy disease free scions was suggested by

Chong et al., (Chong 2005)

Allan et al (Allan 2010) reported the higher percentage of success

(80%) by side grafting obtained after 15 weeks on the vigorous, well fertilized stocks surface sterilized with 10% sodium hypochlorite

Performance of papaya grafts under field condition: Brazilian researchers have experimented with micro-grafting very small seedlings and successfully side grafted plants in the field under tropical conditions (Allan 2009) Grafting papaya on normal dwarfs resulted in plants flowering and producing fruit in the lower stem and with lower productivity compared to papaya ‘Solo’ normal (Senthilkumar 2014) Conversely, grafts of ‘Solo’ dwarf on ‘Solo’ normal, resulted in vigorous plants that flowered first in a higher position on the stem with significant productivity and more uniform distribution of fruits along the stem Ramkhelawan and Baksh (Ramkhelawan 1998) reported at first flowering, the difference in height between seedling plants and those that were terminal wedge grafted, side grafted and chip budded papaya plants were 64.0, 60.9 and 57.9 cm respectively On the other hand, terminal wedge grafted plants had sturdy appearance and were shorter with much larger stem diameters at the base

2.2.2 Cutting propagation

* General of cutting

The propagation of tropical trees by cuttings or stakes is not a new concept and has been done by farmers with a variety of species for centuries, but it has received considerable attention in the scientific community only in the last few decades Propagation by cuttings is a relatively easy method which can provide a large number of plantlets Stem cuttings is the cheapest and most desirable one due to its distinct advantage i.e true to type plants could be raised within shortest period when compared with other vegetative propagation Cutting are the most important means of propagation ornamental shrubs - deciduous species well as the broad- and narrow-leaved types of evergreens Cuttings are also used widely in propagating certain fruit species (Adugna 2015, Alikhani 2011, Cui 2010, Owuor 2009, Paull 2011)

Propagation by cuttings is the method on inducing adventitious root formation of cuttings The types of cutting use in cutting propagation like root cutting, leave bud cutting, leave cutting, stem cutting (Hartmann 2002)

In cutting propagation, it is only necessary that a new adventitious root system be formed, since a potential shoot system is already present

In cutting propagation, there are many factors affecting regeneration of plants from cuttings such as cutting material (Status of the stock plant, rejuvenaton and conditioning of plants prior to cutting, type of wood selected, seasonal timing), how to treatment of cutting, (storage of cutting, growth regulators, mineral nutrition of cutting, leaching of nutrients, fungicides, and wooding), environmental conditional during rooting (water relation, temperature, light, accelerated growth techniques, photosynthesis of cuttings, and rooting medium) (Hartmann 2002)

* Achievements in papaya cutting research

South African horticulturists have worked out a method of propagating papaya (papaws) from cuttings This now means that selected plants can be mass-produced and the variability, fruit size and quality so often encountered

in most papaya plantations can be eliminated by the production of cuttings

Near Pieter Maritzberg, uniform, good-quality papaw fruits, produced on plants propagated vegetatively by cuttings, have brought returns as high as R15,000 per hectare for growers (Newsletter 1982) Plants obtained by cuttings fruited earlier, uniform quality and could be kept in production for a longer time (Allan 2007; Allan 1995)

Allan (Allan 2007) initiated propagation by cuttings of papaya to eliminate the variability in certain clones, so that, their performance could be more accurately compared in evaluation studies These studies were performed with the clones ‘Hortus Gold’ and ‘Honey Gold’, the latter being obtained from the first selections reaching higher standard and is resistant to anthracnose

Many researchers reported that treating the papaya cuttings with IBA applied at the base produced satisfactory results in the induction of roots (Allan 1995, 2007; Fitch 2005; Omar 2016)

Singh et al (Singh 1986) reported that patch budding consistently gave

a higher percentage of success over shield budding The latter is not recommended since the papaya rootstock bark cannot be easily lifted for insertion of bud Vegetative propagation of the superior papaya clone ‘Honey Gold’ through leafy cuttings and grafting has been practiced successfully for over 25 years at South Africa Clones that are adapted to local conditions were selected and propagated vegetatively in other areas

Allan and MacMillan (Allan 1991) had reported on rooting of cuttings

in a mist bed following immersion in a solution of fungicides (2 mg/L dithane and 1 g/L benlate), a 20-min drying period, and a dip in a commercial IBA rooting powder:captan:benlate mix at 9:2:2

Allan et al (Allan 1993) stated that induction and proliferation of

suitable sized lateral shoots for cuttings were improved further with the application of cytokine and gibberellic acid mix The ideal size of cuttings

would be 50-150 mm long and 8-12 mm diameter with 4-5 leaves These are harvested, trimmed to leave 3-4 small leaves and treated with fungicide and a basal dip in IBA to encourage rooting before they were planted in the intermittent mist beds with bottom temperature of 30 oC The cutting would root in about 3 weeks

In South Africa, rooting of cuttings is used to eliminate variability in some papaya cultivars Allan (Allan 1995) and Allan and Carlson (Allan 2007) showed how a female clone ‘Honey Gold’ could be propagated by rooting leafy cuttings for over 40 years These authors stated that vigorous stock plants, strict sanitation, adequate bottom heat (30°C), and even distribution and good control of intermittent mist to ensure leaf retention, are crucial for success Allan and Carlson (Allan 2007) also indicated that suitable rooting media consisted of either perlite or well composted, mature pine bark of varying air-filled porosity (9-30%) and water holding capacity (58-82%) Up to 75-95% rooting of small to medium-sized leafy cuttings could be achieved in six to ten weeks in perlite or mature, composted pine bark during summer, but slow and poor rooting (20% after 16 weeks) occurred in certain bark media The latter was attributed to insufficient bottom heat, different physiological conditions in spring, or toxic compounds other than high levels of tannin Bacterial infection was also regarded as a limiting factor to the success of the procedure

2.2.3 Micropropagation

* General of micropropagation

Research in micropropagation can be traced out in 1965, when French botanist George Morel was attempting to obtain a virus-free orchid plant and discovered that a millimeter-long shoot could be developed into complete plantlets by meristem tip culture Thereafter developed countries began commercial exploitation of this technology, in mid eighties Tissue culture is the ability to establish and maintain plant organs (embryos, shoots, roots, and

flowers) and plant tissues (cell, callus, and protoplasts) in aseptic culture Micropropagation is a form of tissue culture used to regenerate new plant (Hartmann 2002)

Micropropagation is controlled over each aspect of regeneration in tissue culture at level high degree Manipulation in each step of the process is conducted in the tissue culture environment This is the method to propagate plants that are low to multiply or those that cannot be clonally propagated any other way It is also applied to regenerate plants that have been genetically modified though biotechnology Among vegetative propagations, micropropagation is the best method to ensure exclude transmission of systemic diseases from mother plants This method have been applied largely

in many crop (fruit, forest tree, ornamental plants, ect.) (Hartmann 2011)

Plant micropropagation is separated into different stages, each of which

is manipulated by media modification (nutrition, growth regulator, state of medium, etc) and environmental control (light, temperature, air, etc.) There are four distinct stages for most plants: Establishment, multiplication, root formation, and acclimatization (Hartmann 2002)

* Achievements in papaya micropropagation research

Establishment from explants

Shoot tip, axillary bud and single node culture:

Papaya is most commonly propagated by shoot tip or axillary bud (explants around 20 mm in length) in tissue culture This is the most reliable method used for the micropropagation of this fruit tree to date Prior to the collection of shoot tip or axillary bud explants, the mother plant should be tested for the presence of pathogens, in particular viruses and bacteria Virus indexing should be conducted prior to the establishment of cultures and smaller explants are in general recommended Bacterial indexing is also essential, since up to fourteen bacterial isolates can be found in surface-

sterilized shoot tips In one study with shoots of C papaya ‘Surya’, six negative genera, two Gram-positive genera (Thomas 2007, Thomas 2007) were identified Chan and Teo (Chan 1993) used the following method to surface sterilize explants for tissue culture and obtained a 77-84% successful regeneration rate The steps involved; a wash in detergent, then a 30 minute rinse with running tap water; excised apical and axillary buds were placed in 95% ethanol for 15 sec, the surface sterilized for 20 min in 20% chloride (commercial bleach); three rinses with sterile distilled water; immersion in an antibiotic solution containing 100 mg/L chloramphenicol, 100 mg/L streptomycin with continuous agitation on an orbital shaker for 24 h; three rinses with sterile distilled water; incubation in 4% sucrose solution between Whatman № 1 filter paper sheets for 48 h; sterilization for 5 min in 5% chloride (commercial bleach); three rinses with sterile distilled water; plate explants on solid plant growth regulators (PGR)-free MS medium Although this method is effective, it is tedious and time consuming and the use of antibiotics is now be discouraged Fitch (1993) cited that Conover treated papaya explants by immersion in 70% ethanol for 30s and be sterilized in 20% Clorox (commercial product made in Singapore) solution containing 2 drops l-1

Gram-of tween-20 (Sigma, USA) for 20 minutes

Numerous researchers (Rajeevan 1983, Rajeevan 1986, Mondal 1990, Chan 1993, Islam 1993, Lai 2000, Panjaitan 2007, Sanjeev 2013) established

plantlets from both shoot tips and axillary buds Ashmore et al (Ashmore

2001) obtained micro-cuttings from cryopreserved shoot meristems Agnihotri

et al (Agnihotri 2004) could establish male and female plants through shoot

tip culture, but noted much callusing at the base of micro-cuttings

Organogenesis, anther and ovule culture, and regeneration from protoplasts

There is only one single study that reports on the successful regeneration of plants directly from petioles in papaya (Hossain 1993) Litz

and Conover (Litz 1983) reported papaya regeneration by organogenesis from cotyledons of axenically-grown C papaya seedlings Tsay and Su (Tsay

1985) improved the conversion rate (0.7%) when anthers were cultured on

simple medium Rimberia et al (Rimberia 2005) induced somatic embryos

from anthers in a liquid-to-solid 2-phase step using 0.1 mg/L BA and 0.1 mg/L NAA The maximum embryo induction rate increased to 4% when anthers were treated with water for 1 day or MS medium with sucrose for 3 or

5 days These authors then used sex diagnostic PCR to confirm that the plants were female

Ovule culture is limited, and almost exclusively conducted for the

production of somatic embryos Chen et al (Chen 1991) and Chen and Chen

(Chen 1992) were successful to isolate protoplasts from highly regenerable suspension cultures from interspecific crosses of C papaya × C cauliflora zygotic embryos These protoplast- derived somatic embryos proliferated rapidly and some formed plantlets

Callus induction and somatic embryogenesis:

Not all callus tissue induced in papaya is embryogenic, with clusters of meristematic points Once callus with embryogenic potential has been formed

or isolated, it can be maintained effectively using cell suspension cultures Since soma clonal variation and the possible production of off-types is a constant worry, somatic embryogenesis is not a commonly used method for the micropropagation of papaya, even though several positive results have been obtained It remains nonetheless an important method for genetic transformation, as described later in the review

Teixeira et al (Teixeira 2009) cited that De Bruijne et al first induced

somatic embryos from papaya callus using seedling petiole segments but no plants were regenerated in 1974 In contrast, Yie and Liaw used the internode stem of seedlings, first induced callus on MS containing 5.0 μM NAA and 0.5

μM kinetin, then somatic embryos on MS containing 0-0.25 μM IAA and

5.0-10 μM kinetin, and subsequently regenerated plantlets in 1977 And 1978,

Arora and Singh advanced this finding by also inducing roots in vitro from

shoots derived from somatic embryos The authors showed that auxin was critical for the initiation and subsequent growth of callus and that out of the 3 auxins tested, NAA was most effective, followed by 2,4-D and IAA Addition

of 1.0 mg/L NAA was sufficient for good callus growth, occasionally assisted

by the addition of GA3 up to 1.0 mg/L These authors claimed that the milky

latex inhibited the establishment of in vitro cultures from mature tissues of

both male and female plants Litz and Conover (Litz 1982) furthered their own findings by inducing callus from ovules, and somatic embryos that subsequently formed germinated in 10-20% of the cultured ovules both solid and liquid White’s medium supplemented with 60 g l-1 sucrose, 400 mg l-

1glutamine, 20% (v/v) filter-sterilized coconut milk and 8 g l-1 agar Chen et

al (Chen 1987) regenerated somatic embryos in three months from ‘Sunrise Solo’ seedling root explants cultured on ½MS containing 5.4 μM NAA, 2.3

μM kinetin and 2.6 μM GA3, and finally 100 plants per explant Litz and Conover (Litz 1983) induced callus from the midrib (0.3-2.0 mg l-1BA with 0.5-3.0 mg l-1 NAA) and lamina (0.6-3.0 mg l-1 BA with 1.2-5.0 mg l-1NAA)

of cotyledons of axenically-grown C papaya seedlings when cultured on MS

basal medium Fitch (Fitch 1993) induced somatic embryos in ‘Kamiya Solo’ from an initial callus phase when hypocotyl sections were cultured on ½MS with modified MS vitamins, 2.3 to 112.5 mM 2,4-D, 400 mg/L glutamine, and 6% sucrose Yamamoto and Tabata (Yamamoto 1989) also induced hypocotyl somatic embryos using 0.1-1.0 μM 2,4-D Explants of 1 cm were cultured on LS medium containing 10 μM 2,4-D (Yamamoto 1986) Pale yellow, friable embryogenic calli were produced but plantlets were not regenerated since the focus of their studies was on laticifer development in

papaya somatic embryos Monmarson et al (Monmarson 1995) produced

embryogenic calli from the integuments of immature seeds at a frequency Bhattacharya and Khuspe (Bhattacharya 2000) induced somatic embryos in ‘Honey Dew’ and ‘CO2’ following the culture of immature

high-zygotic embryos on MS + 3 mg/L 2,4,5-T in the dark for 3-6 weeks Maturation of embryos was achieved in medium supplemented with ABA at 0.1 mg/L or on PGR-free medium (71% in ‘Honey Dew’ and 59% in ‘CO2’) Romyanon (Romyanon 2007) found that somatic embryos cultured in half-strength liquid MS medium containing 22.5 μM 2,4-D and 2.5 μM ABA yielded higher cell mass (dry-weight basis) than parallel treatments with other combinations of PGRs

Ovules are an excellent source of regenerable papaya cultures via somatic embryogenesis ‘Ovular’ somatic embryos are mainly derived from nucellar tissue (Litz 1982, Litz 1983) Litz and Conover (Litz 1982) also reported that occasionally cultured ovules from self-pollinated papayas also became embryogenic, although the zygotic or maternal origin was not specified Magdalita et al (Magdalita 2002) could achieve 65% somatic embryos from zygotic embryos of ‘Solo’ on de Fossard medium with 0.25

μM each of BAP and NAA and 10.0 μM GA3

Fitch (Fitch 1993) found that an increase in osmoticum up to 7% sucrose resulted in a simultaneous increase in the percentage somatic embryogenesis of ‘Kapoho Solo’ hypocotyls Similar findings were reported

by Litz (Litz 1986)

Genotype also played a role in the success of somatic embryogenesis, with ‘Kapoho’ > ‘Sunset’ > ‘Sunrise’ > ‘Waimanalo’ (Fitch 1993) Fitch and Manshardt (Fitch 1990) had previously found, however that the order was

‘Waimanalo’ > ‘Sunrise’ > ‘Kapoho’ > ‘Sunset’, although this order varied depending on the medium constituents and concentration of phytohormones added For example ‘Waimalo’ showed the lowest (57%) embryogenic yield compared to ‘Sunset’ (93%) when 5 mg/L 2, 4-D was included in the medium In their study, CW, BA, TDZ, 2,4-D or picloram could induce somatic embryos, singly, or in combination Litz and Conover (Litz 1982,

Litz 1983) also found 20% (v/v) CW to be efficient on either MS or White’s medium for the induction of somatic embryos

Jordan et al (Jordan 1983) could induce somatic embryogenesis in

‘mountain papaya’ (C pubescens) using hypocotyl calluses induced from greenhouse- grown seedlings on a medium containing 5-25 μM NAA and 5

μM kinetin

Multiplication

According to Magdalita et al (Magdalita 1997), the accumulation of ethylene in papaya cultures tends to cause an increase in senescence: 3.5-fold higher when the ethylene concentration is 50 ppm as compared to controls These authors reduced ethylene accumulation and senescence by adding a loose cap of aluminum foil and thus increased aeration Other strategies employed by Magdalita et al (1997) to reduce ethylene accumulation and senescence involved the use of larger culture vessels and the inclusion of the ethylene-suppressant aminoethoxyvinylglycine (AVG) at 1.2 μM, or the ethylene-antagonist, silver thiosulphate (STS) at 0.3 mM The use of AVG and STS increased nodal culture growth by 283% and 289%, respectively, while leaf area production was increased by 350% and 211%, respectively Even though nodal culture is an easy technique, involving the sprouting of axillary buds from a node, on suitable medium, this technique is not commonly used

Lai et al (Lai 1998) showed that by aerating shoot buds two weeks

after no aeration gave a 41% increase in the number of shoots ≥ 0.5 cm, a 42% increase in leaf expansion and a 17% increase in leaf numbers compared

to unaerated cultures In independent experiments, Teixeira da Silva (unpublished data) showed how the use of aeration (using a VitronTM vessel

or Milliseal®) and 3000 ppm CO2 photoautotrophic micropropagation could increase the general physiology of papaya in vitro-regenerated plants, including fresh leaf weight and number, and SPAD, i.e measure of

chlorophyll content while the manipulation of the light quality could allow for the formation of “mini”- papaya plantlets through the uses of blue LEDs (light emitting diodes) In 2000, Lai et al (Lai 2000) went further by adding the ethylene biosysntheis precursor, ACC and the inhibitors, AVG and CoCl2, to medium in sealed containers Shoot number was enhanced 75% with the addition of 2 μM ACC and 23% and 49% by the addition of 0.5 μM AVG and

5 μM CoCl2, respectively

Geneve et al (Geneve 2007) showed that pawpaw cultures typically

produce many bud clusters that do not readily elongate and that bud cultures that had been maintained on a BA (8.9 μM) + NAA (2.3 μM) medium for over five years showed evidence of cytokinin habituation Single shoot-buds (1.5 cm) moved to a media with or without PGRs continued to initiate new shoots at a similar rate (~ 5 to 8 shoots per culture)

shoot-Manshardt and Drew (shoot-Manshardt 1998) were able to commercially produce and grow 14,000 elite female clones generated from micro-cuttings

of nodes of apically dominant plants Lai et al (Lai 1998) could mass produce

plants when papaya plantlets were repeatedly subcultured on MS medium supplemented with 0.88 μM BA and 0.1 μM NAA, and this method is currently used to mass produce papaya in Taiwan Similar propagation medium (MSNB) for multiple shoot formation was devised by Yang et al (Yang 1996) in which shoots were induced from petioles on MS supplemented with Gamborg’s B5 vitamins, 0.8 μM BA and 0.1 μM NAA Teo and Chan (Teo 1994) used a 10-week solid proliferation medium followed by a 10-week liquid proliferation medium to mass produce shoots (116 plants per explant) The proliferation medium consisted of MS + 0.1 mg/L BA + 500 mg l-1casein hydrolysate + 0.38 mg l-1 riboflavin Suksa-Ard

et al (Suksa-Ard 1998) showed how elongation of shoot masses, initiated on

an MS medium with BA, could be achieved with the application of 2.5 μM

2-iP on medium containing 3% sucrose and 12 mg l-1agar

Mondal et al (Mondal 1990) used gibberellins to restore apical

dominance following growth on a cytokinin medium, which tends to induce bushiness in vitro

Drew (Drew 1992) found that 1% fructose resulted in better plant production, especially over repeated sub-cultures, than when used 2% sucrose

Rooting and acclimatization

Mass propagation by ex vitro rooting was attempted by Reuveni et al (Reuveni 1990) and Kataoka and Inoue (Kataoka 1992) but stringent rooting conditions, seasonal factors, and explant type affected rooting success (Kataoka 1992, Teo 1994), and thus its application to a mass micropropagation unit

Many papaya tissue culture scientists believe that the addition of an auxin to the medium is an essential prerequisite for successful rooting of in vitro shoots Miller and Drew (Miller 1990) determined the minimum size for

a shoot tip to root is 5 mm, while Drew et al (Drew 1993) claimed a short,

3-day exposure to 10 μM IBA is sufficient to induce roots and that a longer exposure period inhibits root formation These authors also rooted papaya shoots on NAA or CPA supplemented medium Earlier studies by Drew (Drew 1987) showed that when riboflavin was added to the medium, it synergistically acted to promote rooting When Teo and Chan (Chan 1994 ) embedded micro-cuttings on a full or half strength medium (MS) + 2.2 μM

BA + 29.5-49.2 μM IBA, thick, stumpy roots formed with basal callusing To avoid callusing, the same authors suggested a dip in a low agar concentration medium with 12.3 μM IBA Surprisingly, only a 68% success rate was achieved, as opposed to 90% when micro-cuttings were dipped in 11.1 μM IBA and then grown ex vitro in vermiculite

Although early reports of acclimatization claimed poor field performance and survival of papaya (Winnaar 1988), later reports claim a 100% acclimatization success (Drew 1988, Magdalita 1998) Thick, short, stumpy roots and yellowing of leaves have frequently been reported on agar-supplemented medium (Drew 1987, Drew 1989, Drew 1993, Teo 1994, Yu 2000) Suksa-Ard et al (Suksa-Ard 1998) showed how the choice of medium affected the in vitro rooting percentage and demonstrated high rooting rates in starch (96%), then agar (76%), and rockwool (76%) Lower rates were observed with vermiculite (56%) and gellan gum (8%) Agnihotri et al (Agnihotri 2004) could successfully root shoots in a 4-stage process in which the first step involved a 24 h 10 mg/L IBA-pulse

It would appear that the physical and chemical nature of the rooting substrate affects the rooting capacity in papaya considerably (Kataoka 1994)

Yu et al (Yu 2000) established a more efficient protocol in which papaya

shoots were cultured for one week in darkness on MS + 2.5 μM IBA followed

by two weeks in aerated flasks on ½MS, and plantlets acclimatized in vermiculite Resulting survival rates were 94.5% from aerated vermiculite, 87.8% from nonaerated vermiculite, 42.2% from aerated agar, and 35.6% from non-aerated agar Drew (Drew 1988) claimed that a 1:1:1 ratio of peat: perlite: vermiculite provided sufficient aeration to avoid bacterial and fungal

diseases Agnihotri et al (2004), following a rather complex 4-step rooting

process (basically transferring from an IBA-supplemented medium to an IAA-supplemented medium) culminating in a final in vitro rooting step on sterilized Soilrite, demonstrated an 80% survival upon transfer ex vitro

In many coutries, field performance trials of tissue-cultured papaya were conducted (Pandey 1988, Drew 1993, Chan 1994) The results presented that the juvenile period was shortened as compared to plant propagated by sexual, with either earlier flowering, or flowering at a significantly reduced height