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Adventitious shoot formation from sections of sweet potato grown in vitro
Scientia Horticulturae, 20 (1983) 119--129
119
Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
ADVENTITIOUS SHOOT FORMATION FROM SECTIONS OF SWEET P O T A T O GROWN IN V I T R O
LAN SHING HWANG,
ROBERT
M. SKIRVIN, J E A N C A S Y A O
l and J O H N B O U W K A M P
Department of Horticulture, University of Illinois, Urbana, IL (U.S.A.) 1 Philippine Atomic Energy Commission, Diliman, Quezon City, Philippines 3008 2 Department of Horticulture, University of Maryland, College Park, MD, U.S.A. (Accepted for publication 27 September 1982) ABSTRACT
Hwang, L.S., Skirvin, R.M., Casyao, J. and Bouwkamp, J., 1983. Adventitious shoot formation from sections of sweet potato grown in vitro. Scientia Hortic., 20: 119--129. Sweet potato (lpomoea batatas Lam.) root sections were obtained from the cultivars 'Centennial', 'Redmar' and 'Jewel' with a No. 6 cork borer. These sections were cut into 2--3-ram discs and explanted on to modified Murashige and Skoog (MS) medium consisting of MS high mineral salts, myo-inositol (100 mg 1-1), Staba vitamins, 6-benzylaminopurine (2.0 mg l-l), naphthaleneacetic acid (0.1 mg l-l), sucrose (30 g 1-1 ) and agar (10 g l-l). Root discs from internal regions of the tuberous roots gave rise to caUi and meristernatic bud4ike centers (MBLC's). A small percentage of 'Centennial' MBLC's burst open to reveal plantlets which grew and rooted well on the medium. Some of the 'Jewel' MBLC's contained only roots, while those of 'Redmar' did not differentiate. MBLC-formation occurred most often on discs taken from fresh (unstored) roots of 'Centennial'. Petiole sections taken from in vitro-cultured plants of all 3 cultivars developed plants quite readily on the medium. Shoots of all 3 cultivars grew rapidly, to yield whole rooted plants which could easily be moved to soil and grown in the greenhouse and field. Keywords: adventitious shoots; endogenous cycles; Ipomoea batatas; sweet potato; tissue culture; tuberous root; vitro. INTRODUCTION Since s w e e t p o t a t o ( I p o m o e a batatas L a m . ) cultivars are p r o p a g a t e d asexually f r o m a d v e n t i t i o u s s h o o t s (slip's), o n e w o u l d e x p e c t t h e m t o be e x t r e m e l y u n i f o r m . This is n o t t h e case, h o w e v e r , a n d m a n y researchers have r e p o r t e d variability a m o n g hills derived f r o m a single r o o t . T h e p r o b l e m can be so severe, t h a t m a n y cultivars " r u n o u t " after several y e a r s o f c u l t u r e (Wilson et al., 1980). R u n n i n g o u t in sweet p o t a t o does n o t just relate t o disease build-up, b u t m a y be m o r e closely related t o the very h e t e r o g e n o u s genetic m a k e - u p a n d high m u t a t i o n rate. In o r d e r t o m i n i m i z e clonal variability, growers a n d p r o p a g a t o r s have been e n c o u r a g e d either t o use certified s t o c k s or to select seed r o o t s t h a t are relatively t r u e t o t y p e f o r a p a r t i c u l a r cultivar (Wilson et al., 1 9 8 0 ) . 0304-4238/83/$03.00
© 1983 Elsevier Science Publishers B.V.
2
120 Another m e t h o d that might be useful to minimize variability is tissue culture, which is a system whereby cells, tissues or organs can be grown aseptically on artificial media. By changing the composition of media and the concentration of plant growth regulators, it is possible to induce various types of growth. Tissue culture has been utilized to produce sweet potato callus cultures (Bidney and Shepard, 1980) and intact plants from leaf explants (Sehgal, 1975), shoot tips (Litz and Conover, 1978), anthers (Sehgal, 1978; Tsay and Tseng, 1979) and tuberous root segments (Yamaguchi and Nakajima, 1973). However, no careful analysis of the nature of plantlet formation has been made. This study was initiated to develop a reliable m e t h o d to produce sweet potato plants in vitro. MATERIALS
AND METHODS
Three cultivars of sweet potato tuberous roots were obtained from the University of Maryland: 'Centennial', 'Jewel' and 'Redmar'. Some of the tuberous roots were planted in sand and allowed to form rooted shoots (slips) in the greenhouse; other roots were used as explants; and some roots were stored. Shoot tips (1--2 cm long), axillary buds (1--2 buds/section), leaf pieces (0.5 X 0.5 cm) and petiole sections (1 cm long) were taken from shoots. Tissue pieces were excised from each cultivar, surface sterilized with 10% Clorox (0.52% NaOC1), rinsed twice with sterile distilled water, trimmed and then aseptically explanted to culture tubes (25 X 150 mm) containing various media (10 ml). The standard sweet potato medium (SPM) used for these studies was a high mineral salt medium of Murashige and Skoog (1962} containing Staba vitamins, obtained from E.J. Staba, University of Minnesota. A m o u n t s per liter were: cyanocobalmin, 1.5 pg; folic acid, 0.5 mg; riboflavin, 0.5 mg; biotin, 1 mg; choline chloride, 1 mg; Ca pantothenate, 1 mg; thiamine HC1, 1 mg; nicotinamide, 2 mg; pyridoxine HC1, 2 mg; para-amino benzoic acid, 0.5 mg (Skirvin and Chu, 1979). The medium also contained myo-inositol (100 mg l-l), ascorbic acid (50 mg 1-l) and sucrose (30 g l-l). Naphthaleneacetic acid (NAA) (0.1 mg 1-1) and benzyladenine (BA) (2.0 mg 1-l) were added. The pH was adjusted to 5.7 and Difco Bacto agar (6 g 1-1) was also added. The medium was autoclaved at 15 p.s.i, for 20 min. Callus could be grown on this medium if the NAA and BA were replaced with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.5 mg 1-1 } and kinetin (0.1 mg 1-1). Aseptic cultures, which had been established from greenhouse-grown plants, were sometimes utilized as further explant sources. Callused petioles, for instance, were cut into 3 or more pieces and explanted to fresh SPM. Leaf blade and petiole tissue excised from in vitro plants were utilized for a group of experiments to study differentiation. Other sweet potato roots of each cultivar were cleaned by thoroughly
121 brushing the skin (periderm) under running tap-water, removing the bruised or decaying portions of each root, and peeling the roots. Each peridermfree root was then immersed in 10% Clorox for 15 min, washed twice with sterile distilled water, and stored separately in beakers filled with sterile water until t h e y were sectioned. R o o t sections were taken from each root with a No. 6 cork borer (12 mm diameter). The borer was soaked in 70% ethanol for 2 min and then flamed thoroughly. The borer was used to obtain root cylinders both laterally and through the central axis (Fig. 1). Excised cylinders were pushed from the borer with a sterilized glass rod on to sterile Petri dishes. The tissue cylinders were sectioned into 2- to 3-mm slices, which were carefully positioned to maintain their original order within the parent root (Fig. 1). Each slice was put into a culture tube containing 10 ml of medium. The explants were incubated at 23°C with a 16-h day-length and about 2150 lux cool white fluorescent light.
L Fig. 1. Lateral sections o f sweet potato. Central axis cores were obtained by taking cores through the central cylinder.
RESULTS t i p s . - - Shoot tips of each of the 3 cultivars grew, elongated and formed shoots, leaves and roots on the sweet potato medium (SPM).
Shoot
Lateral buds. - - The 1- to 2-bud stem sections also developed into whole plants on SPM. Some of the roots in these cultures enlarged in the a u t u m n
122 ( A u g u s t - O c t o b e r ) to form thickened regions reminiscent of tuberous roots (Fig. 2). Since this p h e n o m e n o n was observed in 2 consecutive years, it seems as if these roots were exhibiting some form of endogenously controlled cycle. The true situation remains unknown.
Fig. 2. The development of enlarged regions on the roots of sweet potato occurred each
August--October. Plants developed from shoot tips and lateral buds were transferred to plastic pots filled with clean quartz sand. They were grown in a room with constant temperature (23°C) and h u m i d i t y for 10--15 days. After they had developed more roots, t h e y were moved to soil in the greenhouse. Some of these plants were grown to m a t u r i t y in the field, where they developed tuberous roots characteristic of their parental cultivar. p i e c e s . - - Leaf pieces taken from intact plants in soil produced abundant callus, but no other growth was observed.
Leaf
123 sections. - - Petiole sections taken from greenhouse-grown plants produced abundant callus, b u t no other growth. Petioles taken from subcultured shoot tips of 'Centennial' and 'Redmar' developed plants in vitro. Callused petiole pieces of 'Redmar' produced only callus, while 'Jewel' petiole pieces formed both callus and shoots.
Petiole
sections. - - R o o t discs from cork-borer sections began to grow almost immediately on SPM. Within 3 weeks, 3 types of growth were present on the surface of r o o t discs, as illustrated in Fig. 3.
Root
Fig. 3. Three types of growth observed on sweet potato root discs in vitro. Light green friable callus (right); dark green, round meristematic bud-like center (middle); and dark green, firm compact callus (left).
Some of the MBLC's eventually developed into shoots or roots. A series of pictures illustrating these phenomena is given in Fig. 4. No growth was normally observed in the discs cut from the end sections of either lateral or central cylinder cores. This was probably due to tissue death, which resulted from Clorox treatment. Also, the rate of contamination was highest in these end pieces. Each of the 3 cultivars grew differently: 'Centennial' gave rise to callus, MBLC's, adventitious shoots and roots; 'Jewel' exhibited callus, MBLC development and r o o t differentiation; and 'Redmar' developed only callus. Since all r o o t discs were handled separately with respect to their original location in a tuberous root, we were able to generalize a b o u t the type of differentiation that one could expect from various regions of a 'Centennial' tuberous root. Both central cylinders and lateral sections exhibited some polarity for the formation of MBLC's, shoots and roots, while Callus forma-
124 tion showed little polarity. In general, the sections which were taken from the most proximal cores produced more MBLC's and shoots than those from distal regions. R o o t formation showed the reverse trend. Lateral sections of 'Centennial' generated significantly more MBLC's than did sections from the central cylinder (Table I).
Fig. 4.
Fig. 4.
tO
127 TABLE I G r o w t h and differentiation of sweet potato root discs f r o m the central cylinder (CC) a n d t h e lateral section (LS) in vitro. Within e a c h cultivar, w i t h i n a c o l u m n , n u m b e r s fol-
lowed by different letters are significantly different at the 5% level by analysis of variance Cultivar
n
% of cultures with:
Callus
MBLC
Shoot
Root
Centennial CC LS
169 221
76 a 62 a
21 a 34 b
2 a 8 a
1 a 0 a
Jewel CC LS
268 364
71 a 63 a
18 a 19 a
0 a 0 a
1a 0 a
Redmar CC LS
185 256
55 a 21 a
0 a 0 a
0 a 0 a
0 a 0 a
We n o t e d t h a t individual sweet potato roots of 'Centennial' and 'Jewel' behaved differently in vitro, and f o u n d t h a t some of the differences among roots could be attributed to the period of time t h a t had expired since they had been harvested. Freshly harvested (unstored) sweet potato roots made more organized structures t h a n tissues from stored roots (Table II). The effect of storage on subsequent in vitro differentiation is a factor to be considered by future researchers. It may be necessary to plan one's experiments such that all treatments can be done with freshly harvested roots. The cause of differences between stored and fresh roots is not clear, but Yamaguchi and Nakajima (1973) have reported that the endogenous cytokinin level in tuberous root tissue plays an important role in adventitious root and shoot formation; the endogenous cytokinin was either increased or activated during storage. High cytokinin concentrations were necessary for adventitious root formation, but even low concentrations were sufficient for adventitious shoot production. Also, the o p t i m u m concentration of cytokinin for organ formation varies with cultivars. Similarly, it is possible that unstored 'Centennial' roots had low cytokinin concentration (optimum) and thus gave rise to more shoots than stored ones.
Fig. 4. In vitro development o f m e r i s t e m a t i c bud-like c e n t e r s (MBLC's) on sweet p o t a t o r o o t discs. A, MBLC a n d friable callus; B, enlarging MBLC in the center o f a disc; C, MBLC f o r m i n g a r o o t ; D, s h o o t e m e r g i n g f r o m MBLC; E, detail o f plant-emergence f r o m MBLC; F, w h o l e p l a n t f r o m MBLC.
128 TA B L E II Effect o f long-term storage (1 year) on growth and differentiation of sweet potato root discs in vitro (central cylinder and lateral sections). Within central cylinder and within lateral sections, within a column, numbers followed by different letters are significantly different at the 5% level by analysis of variance Cultivar
n
% of cultures with: Callus
MBLC
Shoot
Centennial; central cylinder Unstored 42 Stored 120
88 a 75 a
64 a 8b
7a 1b
Centennial; lateral sections Unstored 41 Stored 180
27 a 69 b
32 a 34 a
17 a 6b
Root
0 1
0a 0a
Most of the root discs formed multiple MBLC's on SPM, y e t the rate of shoot emergence from root discs was quite low. There were 622 explants of 'Centennial', but only 23 developed shoots. The problem of low plantproduction was further complicated by the fact that only a single plantlet formed from each disc. It appeared t h a t the first shoot to emerge inhibited the growth of the other MBLC's on the disc. In order to study this dominance effect, we removed some plantlets from MBLC's soon after they emerged. Young plantlets were broken off from their m o t h e r disc and the tissue in the vicinity of the separated plant (MBLC tissue) was excised and transferred to fresh SPM. The m o t h e r disc was dissected further to remove 1 or 2 individual undeveloped MBLC's (isolated MBLC's). The isolated MBLC's, as well as the rest of the m o t h e r disc, were individually transferred to fresh SPM. Two of the MBLC tissue explants produced another plant on SPM. Some of the isolated MBLC's differentiated a small leaf, but none developed into a full plant. Of 11 m o t h e r discs, 2 of t h e m formed a new plantlet in the same area that the first plant had developed and only 1 generated a new plant from a different MBLC. To determine whether the m o t h e r disc was essential for differentiation, we also produced callus which was free of the parental explant. When this callus was transferred to SPM, some cultures formed a hard callus which superficially resembled a MBLC, but no organized growth was ever observed from these structures. In all of our studies, we found that plantlets only developed from meristematic bud-like centers when some parental root tissue was present. It seems t h a t the parental root tissue furnishes some growth substances or nutrients which are essential for MBLC growth and development. Although root discs of both 'Jewel' and ' R e d m a r ' failed to form adventitious shoots in vitro, petiole sections formed plants quite readily. We con-
129 clude, t h e r e f o r e , t h a t scientists i n t e r e s t e d in s w e e t p o t a t o s h o u l d screen m a n y cultivars and m a n y tissues f o r their possible use in tissue c u l t u r e conditions. Tissues o f c h o i c e for a d v e n t i t i o u s b u d f o r m a t i o n are either petiole or t u b e r o u s r o o t sections. ACKNOWLEDGEMENT This research was s u p p o r t e d in p a r t b y t h e University o f Illinois Experim e n t Station.
REFERENCES Bidney, D.L. and Shepard, J.F., 1980. Colony development from sweet potato petiole protoplasts and mesophyll cells. Plant Sci. Lett., 18: 335--342. Litz, R.E. and Conover, R.A., 1978. In vitro propagation of sweet potato. HortScience, 13: 659--660. Mumshige, T. and Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15: 473--497. Sehgal, C.B., 1975. Hormonal control of differentiation in leaf cultures of Ipomoea batatas Poir. Beitr. Biol. Pflanz., 15: 47--52. Sehgal, C.B., 1978. Regeneration of plants from anther culture of sweet potato (Ipomoea batatas Poir.). Z. Pflanzenphysiol.., 88: 349--352. Skirvin, R.M. and Chu, M.C., 1979. In vitro propagation of 'Forever Yours' rose. HortScience, 14: 608--610. Tsay, H.S. and Tseng, M.T., 1979. Embryoid formation and plantlet regeneration from anther callus of sweet potato. Bot. Bull. Acad. Sin., 20: 117--122. Wilson, L.G., Collins, W.W. and Averre, C.W., 1980. Yam alert. North Carolina Agric. Ext. Serv. Circ., AG-196. Yamaguchi, T. and Nakajima, T., 1973. Hormonal regulation of organ formation in cultured tissue derived from root tuber of sweet potato. Proc. 8th Int. Conf. Plant Growth Substances, Tokyo, Japan, pp. 1121--1127.
119
Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
ADVENTITIOUS SHOOT FORMATION FROM SECTIONS OF SWEET P O T A T O GROWN IN V I T R O
LAN SHING HWANG,
ROBERT
M. SKIRVIN, J E A N C A S Y A O
l and J O H N B O U W K A M P
Department of Horticulture, University of Illinois, Urbana, IL (U.S.A.) 1 Philippine Atomic Energy Commission, Diliman, Quezon City, Philippines 3008 2 Department of Horticulture, University of Maryland, College Park, MD, U.S.A. (Accepted for publication 27 September 1982) ABSTRACT
Hwang, L.S., Skirvin, R.M., Casyao, J. and Bouwkamp, J., 1983. Adventitious shoot formation from sections of sweet potato grown in vitro. Scientia Hortic., 20: 119--129. Sweet potato (lpomoea batatas Lam.) root sections were obtained from the cultivars 'Centennial', 'Redmar' and 'Jewel' with a No. 6 cork borer. These sections were cut into 2--3-ram discs and explanted on to modified Murashige and Skoog (MS) medium consisting of MS high mineral salts, myo-inositol (100 mg 1-1), Staba vitamins, 6-benzylaminopurine (2.0 mg l-l), naphthaleneacetic acid (0.1 mg l-l), sucrose (30 g 1-1 ) and agar (10 g l-l). Root discs from internal regions of the tuberous roots gave rise to caUi and meristernatic bud4ike centers (MBLC's). A small percentage of 'Centennial' MBLC's burst open to reveal plantlets which grew and rooted well on the medium. Some of the 'Jewel' MBLC's contained only roots, while those of 'Redmar' did not differentiate. MBLC-formation occurred most often on discs taken from fresh (unstored) roots of 'Centennial'. Petiole sections taken from in vitro-cultured plants of all 3 cultivars developed plants quite readily on the medium. Shoots of all 3 cultivars grew rapidly, to yield whole rooted plants which could easily be moved to soil and grown in the greenhouse and field. Keywords: adventitious shoots; endogenous cycles; Ipomoea batatas; sweet potato; tissue culture; tuberous root; vitro. INTRODUCTION Since s w e e t p o t a t o ( I p o m o e a batatas L a m . ) cultivars are p r o p a g a t e d asexually f r o m a d v e n t i t i o u s s h o o t s (slip's), o n e w o u l d e x p e c t t h e m t o be e x t r e m e l y u n i f o r m . This is n o t t h e case, h o w e v e r , a n d m a n y researchers have r e p o r t e d variability a m o n g hills derived f r o m a single r o o t . T h e p r o b l e m can be so severe, t h a t m a n y cultivars " r u n o u t " after several y e a r s o f c u l t u r e (Wilson et al., 1980). R u n n i n g o u t in sweet p o t a t o does n o t just relate t o disease build-up, b u t m a y be m o r e closely related t o the very h e t e r o g e n o u s genetic m a k e - u p a n d high m u t a t i o n rate. In o r d e r t o m i n i m i z e clonal variability, growers a n d p r o p a g a t o r s have been e n c o u r a g e d either t o use certified s t o c k s or to select seed r o o t s t h a t are relatively t r u e t o t y p e f o r a p a r t i c u l a r cultivar (Wilson et al., 1 9 8 0 ) . 0304-4238/83/$03.00
© 1983 Elsevier Science Publishers B.V.
2
120 Another m e t h o d that might be useful to minimize variability is tissue culture, which is a system whereby cells, tissues or organs can be grown aseptically on artificial media. By changing the composition of media and the concentration of plant growth regulators, it is possible to induce various types of growth. Tissue culture has been utilized to produce sweet potato callus cultures (Bidney and Shepard, 1980) and intact plants from leaf explants (Sehgal, 1975), shoot tips (Litz and Conover, 1978), anthers (Sehgal, 1978; Tsay and Tseng, 1979) and tuberous root segments (Yamaguchi and Nakajima, 1973). However, no careful analysis of the nature of plantlet formation has been made. This study was initiated to develop a reliable m e t h o d to produce sweet potato plants in vitro. MATERIALS
AND METHODS
Three cultivars of sweet potato tuberous roots were obtained from the University of Maryland: 'Centennial', 'Jewel' and 'Redmar'. Some of the tuberous roots were planted in sand and allowed to form rooted shoots (slips) in the greenhouse; other roots were used as explants; and some roots were stored. Shoot tips (1--2 cm long), axillary buds (1--2 buds/section), leaf pieces (0.5 X 0.5 cm) and petiole sections (1 cm long) were taken from shoots. Tissue pieces were excised from each cultivar, surface sterilized with 10% Clorox (0.52% NaOC1), rinsed twice with sterile distilled water, trimmed and then aseptically explanted to culture tubes (25 X 150 mm) containing various media (10 ml). The standard sweet potato medium (SPM) used for these studies was a high mineral salt medium of Murashige and Skoog (1962} containing Staba vitamins, obtained from E.J. Staba, University of Minnesota. A m o u n t s per liter were: cyanocobalmin, 1.5 pg; folic acid, 0.5 mg; riboflavin, 0.5 mg; biotin, 1 mg; choline chloride, 1 mg; Ca pantothenate, 1 mg; thiamine HC1, 1 mg; nicotinamide, 2 mg; pyridoxine HC1, 2 mg; para-amino benzoic acid, 0.5 mg (Skirvin and Chu, 1979). The medium also contained myo-inositol (100 mg l-l), ascorbic acid (50 mg 1-l) and sucrose (30 g l-l). Naphthaleneacetic acid (NAA) (0.1 mg 1-1) and benzyladenine (BA) (2.0 mg 1-l) were added. The pH was adjusted to 5.7 and Difco Bacto agar (6 g 1-1) was also added. The medium was autoclaved at 15 p.s.i, for 20 min. Callus could be grown on this medium if the NAA and BA were replaced with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.5 mg 1-1 } and kinetin (0.1 mg 1-1). Aseptic cultures, which had been established from greenhouse-grown plants, were sometimes utilized as further explant sources. Callused petioles, for instance, were cut into 3 or more pieces and explanted to fresh SPM. Leaf blade and petiole tissue excised from in vitro plants were utilized for a group of experiments to study differentiation. Other sweet potato roots of each cultivar were cleaned by thoroughly
121 brushing the skin (periderm) under running tap-water, removing the bruised or decaying portions of each root, and peeling the roots. Each peridermfree root was then immersed in 10% Clorox for 15 min, washed twice with sterile distilled water, and stored separately in beakers filled with sterile water until t h e y were sectioned. R o o t sections were taken from each root with a No. 6 cork borer (12 mm diameter). The borer was soaked in 70% ethanol for 2 min and then flamed thoroughly. The borer was used to obtain root cylinders both laterally and through the central axis (Fig. 1). Excised cylinders were pushed from the borer with a sterilized glass rod on to sterile Petri dishes. The tissue cylinders were sectioned into 2- to 3-mm slices, which were carefully positioned to maintain their original order within the parent root (Fig. 1). Each slice was put into a culture tube containing 10 ml of medium. The explants were incubated at 23°C with a 16-h day-length and about 2150 lux cool white fluorescent light.
L Fig. 1. Lateral sections o f sweet potato. Central axis cores were obtained by taking cores through the central cylinder.
RESULTS t i p s . - - Shoot tips of each of the 3 cultivars grew, elongated and formed shoots, leaves and roots on the sweet potato medium (SPM).
Shoot
Lateral buds. - - The 1- to 2-bud stem sections also developed into whole plants on SPM. Some of the roots in these cultures enlarged in the a u t u m n
122 ( A u g u s t - O c t o b e r ) to form thickened regions reminiscent of tuberous roots (Fig. 2). Since this p h e n o m e n o n was observed in 2 consecutive years, it seems as if these roots were exhibiting some form of endogenously controlled cycle. The true situation remains unknown.
Fig. 2. The development of enlarged regions on the roots of sweet potato occurred each
August--October. Plants developed from shoot tips and lateral buds were transferred to plastic pots filled with clean quartz sand. They were grown in a room with constant temperature (23°C) and h u m i d i t y for 10--15 days. After they had developed more roots, t h e y were moved to soil in the greenhouse. Some of these plants were grown to m a t u r i t y in the field, where they developed tuberous roots characteristic of their parental cultivar. p i e c e s . - - Leaf pieces taken from intact plants in soil produced abundant callus, but no other growth was observed.
Leaf
123 sections. - - Petiole sections taken from greenhouse-grown plants produced abundant callus, b u t no other growth. Petioles taken from subcultured shoot tips of 'Centennial' and 'Redmar' developed plants in vitro. Callused petiole pieces of 'Redmar' produced only callus, while 'Jewel' petiole pieces formed both callus and shoots.
Petiole
sections. - - R o o t discs from cork-borer sections began to grow almost immediately on SPM. Within 3 weeks, 3 types of growth were present on the surface of r o o t discs, as illustrated in Fig. 3.
Root
Fig. 3. Three types of growth observed on sweet potato root discs in vitro. Light green friable callus (right); dark green, round meristematic bud-like center (middle); and dark green, firm compact callus (left).
Some of the MBLC's eventually developed into shoots or roots. A series of pictures illustrating these phenomena is given in Fig. 4. No growth was normally observed in the discs cut from the end sections of either lateral or central cylinder cores. This was probably due to tissue death, which resulted from Clorox treatment. Also, the rate of contamination was highest in these end pieces. Each of the 3 cultivars grew differently: 'Centennial' gave rise to callus, MBLC's, adventitious shoots and roots; 'Jewel' exhibited callus, MBLC development and r o o t differentiation; and 'Redmar' developed only callus. Since all r o o t discs were handled separately with respect to their original location in a tuberous root, we were able to generalize a b o u t the type of differentiation that one could expect from various regions of a 'Centennial' tuberous root. Both central cylinders and lateral sections exhibited some polarity for the formation of MBLC's, shoots and roots, while Callus forma-
124 tion showed little polarity. In general, the sections which were taken from the most proximal cores produced more MBLC's and shoots than those from distal regions. R o o t formation showed the reverse trend. Lateral sections of 'Centennial' generated significantly more MBLC's than did sections from the central cylinder (Table I).
Fig. 4.
Fig. 4.
tO
127 TABLE I G r o w t h and differentiation of sweet potato root discs f r o m the central cylinder (CC) a n d t h e lateral section (LS) in vitro. Within e a c h cultivar, w i t h i n a c o l u m n , n u m b e r s fol-
lowed by different letters are significantly different at the 5% level by analysis of variance Cultivar
n
% of cultures with:
Callus
MBLC
Shoot
Root
Centennial CC LS
169 221
76 a 62 a
21 a 34 b
2 a 8 a
1 a 0 a
Jewel CC LS
268 364
71 a 63 a
18 a 19 a
0 a 0 a
1a 0 a
Redmar CC LS
185 256
55 a 21 a
0 a 0 a
0 a 0 a
0 a 0 a
We n o t e d t h a t individual sweet potato roots of 'Centennial' and 'Jewel' behaved differently in vitro, and f o u n d t h a t some of the differences among roots could be attributed to the period of time t h a t had expired since they had been harvested. Freshly harvested (unstored) sweet potato roots made more organized structures t h a n tissues from stored roots (Table II). The effect of storage on subsequent in vitro differentiation is a factor to be considered by future researchers. It may be necessary to plan one's experiments such that all treatments can be done with freshly harvested roots. The cause of differences between stored and fresh roots is not clear, but Yamaguchi and Nakajima (1973) have reported that the endogenous cytokinin level in tuberous root tissue plays an important role in adventitious root and shoot formation; the endogenous cytokinin was either increased or activated during storage. High cytokinin concentrations were necessary for adventitious root formation, but even low concentrations were sufficient for adventitious shoot production. Also, the o p t i m u m concentration of cytokinin for organ formation varies with cultivars. Similarly, it is possible that unstored 'Centennial' roots had low cytokinin concentration (optimum) and thus gave rise to more shoots than stored ones.
Fig. 4. In vitro development o f m e r i s t e m a t i c bud-like c e n t e r s (MBLC's) on sweet p o t a t o r o o t discs. A, MBLC a n d friable callus; B, enlarging MBLC in the center o f a disc; C, MBLC f o r m i n g a r o o t ; D, s h o o t e m e r g i n g f r o m MBLC; E, detail o f plant-emergence f r o m MBLC; F, w h o l e p l a n t f r o m MBLC.
128 TA B L E II Effect o f long-term storage (1 year) on growth and differentiation of sweet potato root discs in vitro (central cylinder and lateral sections). Within central cylinder and within lateral sections, within a column, numbers followed by different letters are significantly different at the 5% level by analysis of variance Cultivar
n
% of cultures with: Callus
MBLC
Shoot
Centennial; central cylinder Unstored 42 Stored 120
88 a 75 a
64 a 8b
7a 1b
Centennial; lateral sections Unstored 41 Stored 180
27 a 69 b
32 a 34 a
17 a 6b
Root
0 1
0a 0a
Most of the root discs formed multiple MBLC's on SPM, y e t the rate of shoot emergence from root discs was quite low. There were 622 explants of 'Centennial', but only 23 developed shoots. The problem of low plantproduction was further complicated by the fact that only a single plantlet formed from each disc. It appeared t h a t the first shoot to emerge inhibited the growth of the other MBLC's on the disc. In order to study this dominance effect, we removed some plantlets from MBLC's soon after they emerged. Young plantlets were broken off from their m o t h e r disc and the tissue in the vicinity of the separated plant (MBLC tissue) was excised and transferred to fresh SPM. The m o t h e r disc was dissected further to remove 1 or 2 individual undeveloped MBLC's (isolated MBLC's). The isolated MBLC's, as well as the rest of the m o t h e r disc, were individually transferred to fresh SPM. Two of the MBLC tissue explants produced another plant on SPM. Some of the isolated MBLC's differentiated a small leaf, but none developed into a full plant. Of 11 m o t h e r discs, 2 of t h e m formed a new plantlet in the same area that the first plant had developed and only 1 generated a new plant from a different MBLC. To determine whether the m o t h e r disc was essential for differentiation, we also produced callus which was free of the parental explant. When this callus was transferred to SPM, some cultures formed a hard callus which superficially resembled a MBLC, but no organized growth was ever observed from these structures. In all of our studies, we found that plantlets only developed from meristematic bud-like centers when some parental root tissue was present. It seems t h a t the parental root tissue furnishes some growth substances or nutrients which are essential for MBLC growth and development. Although root discs of both 'Jewel' and ' R e d m a r ' failed to form adventitious shoots in vitro, petiole sections formed plants quite readily. We con-
129 clude, t h e r e f o r e , t h a t scientists i n t e r e s t e d in s w e e t p o t a t o s h o u l d screen m a n y cultivars and m a n y tissues f o r their possible use in tissue c u l t u r e conditions. Tissues o f c h o i c e for a d v e n t i t i o u s b u d f o r m a t i o n are either petiole or t u b e r o u s r o o t sections. ACKNOWLEDGEMENT This research was s u p p o r t e d in p a r t b y t h e University o f Illinois Experim e n t Station.
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